Uncovering a unique pathogenic mechanism of SARS-CoV-2 omicron variant: selective induction of cellular senescence.

BACKGROUND: SARS-CoV-2 variants are constantly emerging with a variety of changes in the conformation of the spike protein, resulting in alterations of virus entry mechanisms. Solely omicron variants use the endosomal clathrin-mediated entry. Here, we investigate the influence of defined altered spike formations to study their impact on premature cellular senescence. METHODS: In our study, in vitro infections of SARS-CoV-2 variants delta (B.1.617.2) and omicron (B.1.1.529) were analyzed by using human primary small alveolar epithelial cells and human ex vivo lung slices. We confirmed cellular senescence in human lungs of COVID-19 patients. Hence, global gene expression patterns of infected human primary alveolar epithelial cells were identified via mRNA sequencing. RESULTS: Solely omicron variants of SARS-CoV-2 influenced the expression of cell cycle genes, highlighted by an increased p21 expression in human primary lung cells and human ex vivo lungs. Additionally, an upregulated senescence-associated secretory phenotype (SASP) was detected. Transcriptomic data indicate an increased gene expression of p16, and p38 in omicron-infected lung cells. CONCLUSIONS: Significant changes due to different SARS-CoV-2 infections in human primary alveolar epithelial cells with an overall impact on premature aging could be identified. A substantially different cellular response with an upregulation of cell cycle, inflammation- and integrin-associated pathways in omicron infected cells indicates premature cellular senescence.

Influenza Virus-Induced Paracrine Cellular Senescence of the Lung Contributes to Enhanced Viral Load.

Aging is a major risk factor associated with increased morbidity and mortality rates observed during respiratory infections. In this study, we investigated the role of influenza virus infections in the establishment of premature cellular senescence and paracrine macrophage-activated inflammation. We observed in our murine model a premature aging by the appearance of senescent cells in the lungs after 21 d of influenza A virus infection. By using murine ex vivo lung models, the influence of TNF-α on the establishment of cellular senescence was detectable. Our findings were proven by using conditioned media of infected human monocyte-derived macrophages on primary lung fibroblasts. Here, a distinct expression of senescence-associated parameters could be confirmed. Furthermore, senescent cells in the lungs strongly influenced subsequent viral infections. Our data demonstrated a higher viral load in senescent primary lung fibroblasts, indicating an intracellular effect on viral replication. Transcriptomic data revealed an increased regulation of JAK/STAT signaling in senescent IAV-infected cells accompanied with increased TRAIL expression. Additionally, senescent cells indicating low pH values, accelerating viral replication. Our study provides new insights into pathomechanisms of virus-induced cellular senescence. Hence, IAV infection induces premature senescence and subsequent infections in senescent cells lead to an increased viral replication.

Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway.

The decision whether endosomes enter the degradative or recycling pathway in mammalian cells is of fundamental importance for pathogen killing, and its malfunctioning has pathological consequences. We discovered that human p11 is a critical factor for this decision. The HscA protein present on the conidial surface of the human-pathogenic fungus Aspergillus fumigatus anchors p11 on conidia-containing phagosomes (PSs), excludes the PS maturation mediator Rab7, and triggers binding of exocytosis mediators Rab11 and Sec15. This reprogramming redirects PSs to the non-degradative pathway, allowing A. fumigatus to escape cells by outgrowth and expulsion as well as transfer of conidia between cells. The clinical relevance is supported by the identification of a single nucleotide polymorphism in the non-coding region of the S100A10 (p11) gene that affects mRNA and protein expression in response to A. fumigatus and is associated with protection against invasive pulmonary aspergillosis. These findings reveal the role of p11 in mediating fungal PS evasion.

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis.

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.

Replication in the Mononuclear Phagocyte System (MPS) as a Determinant of Hantavirus Pathogenicity.

Members of different virus families including Hantaviridae cause viral hemorrhagic fevers (VHFs). The decisive determinants of hantavirus-associated pathogenicity are still enigmatic. Pathogenic hantavirus species, such as Puumala virus (PUUV), Hantaan virus (HTNV), Dobrava-Belgrade virus (DOBV), and Sin Nombre virus (SNV), are associated with significant case fatality rates. In contrast, Tula virus (TULV) only sporadically causes mild disease in immunocompetent humans and Prospect Hill virus (PHV) so far has not been associated with any symptoms. They are thus defined here as low pathogenic/apathogenic hantavirus species. We found that productive infection of cells of the mononuclear phagocyte system (MPS), such as monocytes and dendritic cells (DCs), correlated well with the pathogenicity of hantavirus species tested. HTNV (intermediate case fatality rates) replicated more efficiently than PUUV (low case fatality rates) in myeloid cells, whereas low pathogenic/apathogenic hantavirus species did not produce any detectable virus titers. Analysis of PHPUV, a reassortant hantavirus derived from a pathogenic (PUUV) and an apathogenic (PHV) hantavirus species, indicated that the viral glycoproteins are not decisive for replication in MPS cells. Moreover, blocking acidification of endosomes with chloroquine decreased the number of TULV genomes in myeloid cells suggesting a post-entry block for low pathogenic/apathogenic hantavirus species in myeloid cells. Intriguingly, pathogenic but not low pathogenic/apathogenic hantavirus species induced conversion of monocytes into inflammatory DCs. The proinflammatory programming of MPS cells by pathogenic hantavirus species required integrin signaling and viral replication. Our findings indicate that the capacity to replicate in MPS cells is a prominent feature of hantaviral pathogenicity.

Analysis of the integrin ß3 receptor for pathogenic orthohantaviruses in rodent host species.

Host reservoir specificity of pathogens is complex and may depend on receptor variability. For pathogenic orthohantaviruses, integrin ß3 had been previously identified as entry receptor and the presence of aspartic acid residue at position 39 (D39) in human integrin ß3 was described to be a prerequisite for infection of primate cells with Hantaan virus (HTNV). However, the role of integrin ß3 in orthohantavirus infection of host animals is not completely understood. Therefore, we analyzed the nucleotide sequence of the integrin ß3 gene of Myodes glareolus and Apodemus agrarius, the hosts of Puumala virus (PUUV) and HTNV, respectively. Sequence analysis in tissue samples demonstrated that the amino acid residue D39 is not present in integrin ß3 of these natural orthohantavirus hosts. Furthermore, we analyzed the transcription and protein expression levels of integrin ß3 in the renal cell line BVK168 generated from the PUUV host, bank vole. Transcription level of integrin ß3 was 100-fold lower in BVK168 cells than in Vero E6 cells and integrin ß3 expression was not detectable in BVK168 cells. However, despite the absence of amino acid residue D39 and no detectable integrin ß3 expression, BVK168 cells are susceptible to infection with both PUUV and HTNV. These results indicate that the mechanism of orthohantaviral entry in rodent species does not correspond to the requirements that were described for the entry in primate cells in vitro.

Phylogenetic analysis of Puumala virus strains from Central Europe highlights the need for a full-genome perspective on hantavirus evolution.

Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is the medically most important hantavirus in Central and Western Europe. In this study, a total of 523 bank voles (408 from Germany, 72 from Slovakia, and 43 from Czech Republic) collected between the years 2007-2012 were analyzed for the presence of hantavirus RNA. Partial PUUV genome segment sequences were obtained from 51 voles. Phylogenetic analyses of all three genome segments showed that the newfound strains cluster with other Central and Western European PUUV strains. The new sequences from Sumava (Bohemian Forest), Czech Republic, are most closely related to the strains from the neighboring Bavarian Forest, a known hantavirus disease outbreak region. Interestingly, the Slovak strains clustered with the sequences from Bohemian and Bavarian Forests only in the M but not S segment analyses. This well-supported topological incongruence suggests a segment reassortment event or, as we analyzed only partial sequences, homologous recombination. Our data highlight the necessity of sequencing all three hantavirus genome segments and of a broader bank vole screening not only in recognized endemic foci but also in regions with no reported human hantavirus disease cases.

Adler hantavirus, a new genetic variant of Tula virus identified in Major's pine voles (Microtus majori) sampled in southern European Russia.

Although at least 30 novel hantaviruses have been recently discovered in novel hosts such as shrews, moles and even bats, hantaviruses (family Bunyaviridae, genus Hantavirus) are primarily known as rodent-borne human pathogens. Here we report on identification of a novel hantavirus variant associated with a rodent host, Major's pine vole (Microtus majori). Altogether 36 hantavirus PCR-positive Major's pine voles were identified in the Krasnodar region of southern European Russia within the years 2008-2011. Initial partial L-segment sequence analysis revealed novel hantavirus sequences. Moreover, we found a single common vole (Microtusarvalis) infected with Tula virus (TULV). Complete S- and M-segment coding sequences were determined from 11 Major's pine voles originating from 8 trapping sites and subjected to phylogenetic analyses. The data obtained show that Major's pine vole is a newly recognized hantavirus reservoir host. The newfound virus, provisionally called Adler hantavirus (ADLV), is closely related to TULV. Based on amino acid differences to TULV (5.6-8.2% for nucleocapsid protein, 9.4-9.5% for glycoprotein precursor) we propose to consider ADLV as a genotype of TULV. Occurrence of ADLV and TULV in the same region suggests that ADLV is not only a geographical variant of TULV but a host-specific genotype. High intra-cluster nucleotide sequence variability (up to 18%) and geographic clustering indicate long-term presence of the virus in this region.

Detection of shrew-borne hantavirus in Eurasian pygmy shrew (Sorex minutus) in Central Europe.

Recently, it was found that not only rodents but also shrews are reservoir hosts of hantaviruses. In Central Europe, only Seewis virus, associated with the Eurasian common shrew (Sorex araneus), has been recognized until now. In the present report, tissue samples from shrews belonging to Crocidurinae and Soricinae subfamilies, trapped in Czech Republic, Germany, and Slovakia, were screened for the presence of novel hantaviruses. Three new hantavirus partial L-segment sequences were obtained from pygmy shrews (Sorex minutus) trapped in Czech Republic and Germany. Complete nucleocapsid protein- and glycoprotein precursor-coding S- and M-segment sequences were then determined for the newly recognized hantavirus strains, CZ/Beskydy/412/2010/Sm, CZ/Drahany/420/2010/Sm, and DE/Dürrbach/1912/2009/Sm. Phylogenetic analyses showed that they represent strains of Asikkala virus (ASIV), a novel hantavirus also found in pygmy shrews from Finland. Our study reveals a broad geographic distribution of ASIV across Europe and indicates pygmy shrew as the primary reservoir host. Future studies will have to determine the pathogenic relevance of ASIV.

Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe.

For a long time hantaviruses were believed to be exclusively rodent-borne pathogens. Recent findings of numerous shrew- and mole-borne hantaviruses raise important questions on their phylogenetic origin. The objective of our study was to prove the presence and distribution of shrew-associated Seewis virus (SWSV) in different Sorex species in Central Europe. Therefore, a total of 353 Sorex araneus, 59 S. minutus, 27 S. coronatus, and one S. alpinus were collected in Germany, the Czech Republic, and Slovakia. Screening by hantavirus-specific L-segment RT-PCR revealed specific amplification products in tissues of 49 out of 353 S. araneus and four out of 59 S. minutus. S-segment sequences were obtained for 45 of the L-segment positive S. araneus and all four L-segment positive S. minutus. Phylogenetic investigation of these sequences from Germany, the Czech Republic, and Slovakia demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria, and other sites in Germany. The low intra-cluster sequence variability and the high inter-cluster divergence suggest a long-term SWSV evolution in isolated Sorex populations. In 28 of the 49 SWSV S-segment sequences, an additional putative open reading frame (ORF) on the opposite strand to the nucleocapsid protein-encoding ORF was identified. This is the first comprehensive sequence analysis of SWSV strains from Germany, the Czech Republic, and Slovakia, indicating its broad geographical distribution and high genetic divergence. Future studies have to prove whether both S. araneus and S. minutus represent SWSV reservoir hosts or spillover infections are responsible for the parallel molecular detection of SWSV in both species.