Detection of herpes simplex virus Type-1 in patients with fibrotic lung diseases

The current study intends to investigate i) the incidence of herpes viruses including Herpes Simplex Virus type-1 (HSV-1), Cytomegalovirus (CMV) and Human Herpes Virus -6, -7, -8 (HHV6, HHV7, HHV8) in two biological samples, bronchoalveolar lavage fluid (BALF) and lung tissue biopsy, in different forms of pulmonary fibrosis, and ii) the induction of molecular pathways involved in fibrosis by herpesvirus infection in primary cell cultures. PCR was employed for the detection of CMV, HHV6-8 and HSV-1 DNA in lung specimens (4 controls and 11 IPF specimens) and BALF pellet [6 controls and 20 fibrotic Idiopathic Intestitial Pneumonias (f-IIPs) samples: 13 idiopathic pulmonary fibrosis (IPF) and 7 nonspecific idiopathic interstitial pneumonia (NSIP)] samples. Among all herpesviruses tested, HSV-1 was detected in 1/11 (9%) specimens from IPF lung tissue and in 2/20 (10%) samples of f-IIPs BALF whereas the control group was negative. Primary cell cultures from BALF of patients with IPF and healthy controls were infected in vitro with wild-type HSV-1 virus and Real Time PCR was employed for the detection of gene transcription of specific axes implicated in lung fibrosis. Primary cell cultures were permissive to HSV-1, resulting in an upregulation of the fibrotic growth factors TGFβ1 and FGF, the angiogenetic markers SDF1a, SDF1b, VEGF, FGF and the regulators of tissue wound healing MMP9 and CCR7. Downregulation was noted for the CXCR4 and MMP2 genes, while a different response has been detected in healthy donors regarding the expression of the aforementioned markers. These results implicate for the first time the HSV-1 with Fibrotic Idiopathic Interstitial Pneumonias since the virus presented similar incidence in two different biological samples

Self-eating: Friend or foe? The emerging role of autophagy in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is the most common and severe form of idiopathic interstitial pneumonias. Despite an exponential increase in our understanding of potentially important mediators and mechanisms, the pathogenesis remains elusive, and little therapeutic progress has been made in the last few years. Mortality in 3-5 years is still 50%. Autophagy, a highly conserved homeostatic mechanism necessary for cell survival, has been recently implicated in the pathogenesis of pulmonary disorders. In this paper we aim to highlight some key issues regarding the process of autophagy and its possible association with the pathogenesis of idiopathic pulmonary fibrosis

p-cymene impairs SARS-CoV-2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS-CoV-2 nucleocapsid protein and H1N1 nucleoprotein

Therapeutic regimens for the COVID-19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS-CoV-2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p-cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS-CoV-2-induced COVID-19 and other RNA-virus-induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS-CoV-2 C-terminal structured domain contains a nuclear localization signal (NLS), like SARS-CoV, on which p-cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA-viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p-cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p-cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS-CoV-2 to influenza A infections

SARS-CoV-2 Molecular Transmission Clusters and Containment Measures in Ten European Regions during the First Pandemic Wave

International audienceBackground: The spatiotemporal profiling of molecular transmission clusters (MTCs) using viral genomic data can effectively identify transmission networks in order to inform public health actions targeting SARS-CoV-2 spread. Methods: We used whole genome SARS-CoV-2 sequences derived from ten European regions belonging to eight countries to perform phylogenetic and phylodynamic analysis. We developed dedicated bioinformatics pipelines to identify regional MTCs and to assess demographic factors potentially associated with their formation. Results: The total number and the scale of MTCs varied from small household clusters identified in all regions, to a super-spreading event found in Uusimaa-FI. Specific age groups were more likely to belong to MTCs in different regions. The clustered sequences referring to the age groups 50–100 years old (y.o.) were increased in all regions two weeks after the establishment of the lockdown, while those referring to the age group 0–19 y.o. decreased only in those regions where schools’ closure was combined with a lockdown. Conclusions: The spatiotemporal profiling of the SARS-CoV-2 MTCs can be a useful tool to monitor the effectiveness of the interventions and to reveal cryptic transmissions that have not been identified through contact tracing

SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients

Visualization of parental HSV-1 genomes and replication compartments in association with ND10 in live infected cells

The relative location of active and repressed genes within the nucleus is becoming recognized as a significant factor in the control of gene expression. We have developed systems to visualize parental and replicated herpes simplex virus type 1 (HSV-1) amplicon genomes in association with PML nuclear bodies (ND10) in live cells. Plasmids containing viral replication and packaging signals, a gene expressing enhanced yellow fluorescent protein linked to the tetracycline repressor DNA binding domain and 14 copies of the tetracycline operator sequence were used to produce amplicon genomes packaged into normal viral particles. The frequency of the juxtaposition of viral genomes and ND10 was substantially increased by inclusion of an active HSV-1 Early gene transcription unit, indicating that the association is neither random nor passive. Furthermore, the ND10-associated genomes preferentially progressed to form viral replication compartments. Thus, active viral transcription contributes to the efficiency of viral genome association with ND10, and this in turn increases the probability that the genome will engage in active DNA replication. These studies in live cells provide a novel insight into virus–ND10 interactions and provide compelling visualization of their functional relevance

Merkel Cell Polyomavirus (MCPyV) and Cancers: Emergency Bell or False Alarm?

Merkel cell polyomavirus (MCPyV), the sole member of Polyomavirus associated with oncogenesis in humans, is the major causative factor of Merkel cell carcinoma (MCC), a rare, neuroendocrine neoplasia of the skin. Many aspects of MCPyV biology and oncogenic mechanisms remain poorly understood. However, it has been established that oncogenic transformation is the outcome of the integration of the viral genome into the host DNA. The high prevalence of MCPyV in the population, along with the detection of the virus in various human tissue samples and the strong association of MCPyV with the emergence of MCC, have prompted researchers to further investigate the role of MCPyV in malignancies other than MCC. MCPyV DNA has been detected in several different non-MCC tumour tissues but with significantly lower prevalence, viral load and protein expression. Moreover, the two hallmarks of MCPyV MCC have rarely been investigated and the studies have produced generally inconsistent results. Therefore, the outcomes of the studies are inadequate and unable to clearly demonstrate a direct correlation between cellular transformation and MCPyV. This review aims to present a comprehensive recapitulation of the available literature regarding the association of MCPyV with oncogenesis (MCC and non-MCC tumours)

Differential expression and mutation of the ras family genes in human breast cancer

The expression of ras mRNA levels in 27 human sporadic breast cancer specimens was examined, and compared to the corresponding adjacent normal tissue using the RT-PCR technique. Eighteen out of the 27 specimens (67%) exhibited two- to four-fold increased expression of ras mRNA levels, compared to corresponding normal tissue. The rates of augmented mRNA expression were similar among the three ras genes. A statistically significant correlation of overexpression of ras genes in specimens classified as Stage I disease was observed, compared to tumors in a more advanced stage (II or III). The incidence of codon 12 point mutations of the K-ras gene in fresh tissue samples was also assessed in 61 human sporadic breast cancer cases. Point mutations were detected in four (6.5%) out of the 61 cases examined; no correlation was found with any clinicopathological parameter. This is the first report to our knowledge of the differential expression of the ras family genes in breast carcinoma. Our findings indicate that the aberrant expression of ras genes may be an initial event in breast cancer oncogenesis and that K-ras point mutations are rarely involved in the development of mammary neoplasias