The influenza A virus promotes fungal growth of Aspergillus fumigatus via direct interaction in vitro.

Seasonal influenza A virus (IAV) infections still pose a major burden for public health worldwide. Severe disease progression or even death is often related to superinfections of the virus and a secondary bacterial pathogen. However, fungi, especially Aspergillus fumigatus, are also frequently diagnosed during IAV infection. Although, clinical studies have reported the severity of influenza-associated pulmonary aspergillosis, the molecular mechanisms underlying this type of disease are poorly understood. Here, a new in vitro model is introduced that allows the investigation of complex pathogen-host and pathogen-pathogen interactions during coinfection of lung epithelial cells with IAV and A. fumigatus. Our data reveal a reduced IAV load and IAV-induced cytokine and chemokine expression in the presence of A. fumigatus. At the same time, IAV infection promotes the growth of A. fumigatus. Even in the absence of the human host cell, purified IAV particles are able to induce hyphal growth, due to a direct interaction of the virus particles with the fungal surface. Thus, our study gives first insights into the complex interplay between IAV, A. fumigatus and the host cell as well as the two pathogens alone.

Exploring the Diversity of Plant-Associated Viruses and Related Viruses in Riverine Freshwater Samples Collected in Berlin, Germany.

Plant-infecting RNA viruses from 30 families and floating genera, as well as a great number of uncultured as yet-unclassified plant-associated viruses have been described. Even so, the plant RNA virosphere is still underexplored. RNA extracted from enriched virus particles of 50 L water samples from the Teltow Canal and the Havel River in Berlin, Germany, was sequenced using Illumina next-generation sequencing. Sequences were searched for plant viruses with BLAST and DIAMOND. Phylogenetic analyses were conducted with IQ-TREE 2. Altogether, 647 virus sequences greater than 1 kb were detected and further analyzed. These data revealed the presence of accepted and novel viruses related to Albetovirus, Alphaflexiviridae, Aspiviridae, Bromoviridae, Endornaviridae, Partitiviridae, Potyviridae, Solemoviridae, Tombusviridae and Virgaviridae. The vast majority of the sequences were novel and could not be taxonomically assigned. Several tombus- and endorna-like viruses make use of alternative translation tables that suggest unicellular green algae, ciliates, or diplomonades as their hosts. The identification of 27 albeto-like satellite viruses increases available sequence data five-fold. Sixteen new poty-like viruses align with other poty-like viruses in a link that combines the Astroviridae and Potyviridae families. Further, the identification of viruses with peptidase A6-like and peptidase A21-like capsid proteins suggests horizontal gene transfer in the evolution of these viruses.

Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed Ulva.

Marine macroalgae (seaweeds) are important primary producers and foundation species in coastal ecosystems around the world. Seaweeds currently contribute to an estimated 51% of the global mariculture production, with a long-term growth rate of 6% per year, and an estimated market value of more than US$11.3 billion. Viral infections could have a substantial impact on the ecology and aquaculture of seaweeds, but surprisingly little is known about virus diversity in macroalgal hosts. Using metagenomic sequencing, we characterized viral communities associated with healthy and bleached specimens of the commercially important green seaweed Ulva. We identified 20 putative new and divergent viruses, of which the majority belonged to the Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses [single-stranded (ss)DNA genomes], Durnavirales [double-stranded (ds)RNA], and Picornavirales (ssRNA). Other newly identified RNA viruses were related to the Ghabrivirales, the Mitoviridae, and the Tombusviridae. Bleached Ulva samples contained particularly high viral read numbers. While reads matching assembled CRESS DNA viruses and picorna-like viruses were nearly absent from the healthy Ulva samples (confirmed by qPCR), they were very abundant in the bleached specimens. Therefore, bleaching in Ulva could be caused by one or a combination of the identified viruses but may also be the result of another causative agent or abiotic stress, with the viruses simply proliferating in already unhealthy seaweed tissue. This study highlights how little we know about the diversity and ecology of seaweed viruses, especially in relation to the health and diseases of the algal host, and emphasizes the need to better characterize the algal virosphere. IMPORTANCE Green seaweeds of the genus Ulva are considered a model system to study microbial interactions with the algal host. Remarkably little is known, however, about viral communities associated with green seaweeds, especially in relation to the health of the host. In this study, we characterized the viral communities associated with healthy and bleached Ulva. Our findings revealed the presence of 20 putative novel viruses associated with Ulva, encompassing both DNA and RNA viruses. The majority of these viruses were found to be especially abundant in bleached Ulva specimens. This is the first step toward understanding the role of viruses in the ecology and aquaculture of this green seaweed.

Hepeliviruses in two waterbodies in Berlin, Germany.

The order Hepelivirales comprises RNA viruses of four families (Alphatetraviridae, Benyviridae, Hepeviridae, and Matonaviridae). Sequencing of virus genomes from water samples from the Havel River and the Teltow Canal (Teltowkanal) in Berlin, Germany, revealed 25 almost complete and 68 partial genomes of viruses presumably belonging to the order Hepelivirales. Only one of these viruses exhibited a relationship to a known member of this order. The members of one virus clade have a polymerase with a permuted order of the conserved palm subdomain motifs resembling the polymerases of permutotetraviruses and birnaviruses. Overall, our study further demonstrates the diversity of hepeliviruses and indicates the enzootic prevalence of hepeliviruses in unknown hosts.

Picorna-Like Viruses of the Havel River, Germany.

To improve the understanding of the virome diversity of riverine ecosystems in metropolitan areas, a metagenome analysis was performed with water collected in June 2018 from the river Havel in Berlin, Germany. After enrichment of virus particles and RNA extraction, paired-end Illumina sequencing was conducted and assignment to virus groups and families was performed. This paper focuses on picorna-like viruses, the most diverse and abundant group of viruses with impact on human, animal, and environmental health. Here, we describe altogether 166 viral sequences ranging in size from 1 to 11.5 kb. The 71 almost complete genomes are comprised of one candidate iflavirus, one picornavirus, two polycipiviruses, 27 marnaviruses, 27 dicistro-like viruses, and 13 untypeable viruses. Many partial picorna-like virus sequences up to 10.2 kb were also investigated. The sequences of the Havel picorna-like viruses represent genomes of seven of eight so far known Picornavirales families. Detection of numerous distantly related dicistroviruses suggests the existence of additional, yet unexplored virus groups with dicistronic genomes, including few viruses with unusual genome layout. Of special interest is a clade of dicistronic viruses with capsid protein-encoding sequences at the 5'-end of the genome. Also, monocistronic viruses with similarity of their polymerase and capsid proteins to those of dicistroviruses are interesting. A second protein with NTP-binding site present in the polyprotein of solinviviruses and related viruses needs further attention. The results underline the importance to study the viromes of fluvial ecosystems. So far acknowledged marnaviruses have been isolated from marine organisms. However, the present study and available sequence data suggest that rivers and limnic habitats are relevant ecosystems with circulation of marnaviruses as well as a plethora of unknown picorna-like viruses.

A proposed division of the family Picornaviridae into subfamilies based on phylogenetic relationships and functional genomic organization.

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.

Antiviral susceptibility of recombinant Herpes simplex virus 1 strains with specific polymerase amino acid changes.

Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration, however, can lead to the emergence of resistant strains with altered viral thymidine kinase (TK) function, especially in immunocompromised patients. Furthermore, amino acid (aa) changes of the viral deoxyribonucleic acid polymerase (POL) may contribute to resistance to the aforementioned nucleoside analogues. Given this, treatment with foscarnet (FOS) or cidofovir (CDV) may represent an important alternative. Both drugs directly affect POL activity. Several aa changes of POL, such as L49I, E70K, L359I, E421V, P829S, T1121M, and M1226I, have been observed in ACV-resistant clinical strains which also carried relevant aa changes in their TK. Their contribution to ACV, FOS, and CDV resistance is not fully understood. In this study, these seven aa changes with unknown significance for ACV, FOS and CDV resistance were introduced separately into the POL of a recombinant HSV-1 strain rHSV-1(17+)Lox, equipped with or without information for expression of green fluorescent protein (GFP). The GFP-expressing variants were tested for susceptibility to ACV, FOS and CDV. An rHSV-1(17+)Lox GFP strain with the S724N change conferring resistance to ACV and FOS was generated and included as a control. Only the S724N change was confirmed to induce ACV and FOS resistance, whereas the other changes did not contribute to resistance. The underlying nucleotide substitutions of the POL gene should be therefore considered as natural polymorphism. These data will improve sequence-based prediction of antiviral susceptibility.

A novel dicistrovirus in a captive red squirrel (Sciurus vulgaris).

Dicistroviruses are single-stranded RNA viruses in the family Dicistroviridae. The viruses have mainly been detected in arthropods and are the cause of several devastating diseases in many of these species such as honeybees. Increasingly, dicistroviruses have also been detected in both mammalian and avian species in faeces, blood and liver, but with unconfirmed pathology. Here, we report a novel dicistrovirus detected in the intestinal content of a captive red squirrel with enteritis along with the disease history, pathology and genomic characterisation of the virus. Virus particle morphology resembled those of picornaviruses with a diameter of 28-32 nm but failed to be detected using a mammalian/avian pan viral microarray. Next-generation sequencing confirmed a dicistrovirus having a typical dicistrovirus genome organization, but with the polyprotein 1 being shorter by about 100 amino acids, compared to that of other dicistroviruses. Phylogenetic analysis of ORF1 and ORF2 sequences clustered the virus with two yet unassigned dicistroviruses detected in Gorilla gorilla and a freshwater arthropod and likely to be designated to a new genus. Our data further highlights the ever-growing diversity of dicistroviruses, but the clinical significance of the virus in mammalian species and particularly red squirrels has yet to be established.

SARS-CoV-2 causes severe epithelial inflammation and barrier dysfunction.

Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.

Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue.

In rheumatoid arthritis (RA), the expression of many pro-destructive/pro-inflammatory proteins depends on the transcription factor AP-1. Therefore, our aim was to analyze the presence and functional relevance of mutations in the coding regions of the AP-1 subunits of the fos and jun family in peripheral blood (PB) and synovial membranes (SM) of RA and osteoarthritis patients (OA, disease control), as well as normal controls (NC). Using the non-isotopic RNAse cleavage assay, one known polymorphism (T252C: silent; rs1046117; present in RA, OA, and NC) and three novel germline mutations of the cfos gene were detected: (i) C361G/A367G: Gln121Glu/Ile123Val, denoted as "fos121/123"; present only in one OA sample; (ii) G374A: Arg125Lys, "fos125"; and (iii) C217A/G374A: Leu73Met/Arg125Lys, "fos73/125", the latter two exclusively present in RA. In addition, three novel somatic cjun mutations (604-606ΔCAG: ΔGln202, "jun202"; C706T: Pro236Ser, "jun236"; G750A: silent) were found exclusively in the RA SM. Tansgenic expression of fos125 and fos73/125 mutants in NIH-3T3 cells induced an activation of reporter constructs containing either the MMP-1 (matrix metalloproteinase) promoter (3- and 4-fold, respectively) or a pentameric AP-1 site (approximately 5-fold). Combined expression of these two cfos mutants with cjun wildtype or mutants (jun202, jun236) further enhanced reporter expression of the pentameric AP-1 construct. Finally, genotyping for the novel functionally relevant germline mutations in 298 RA, 288 OA, and 484 NC samples revealed no association with RA. Thus, functional cfos/cjun mutants may contribute to local joint inflammation/destruction in selected patients with RA by altering the transactivation capacity of AP-1 complexes.

Laser spectroscopic technique for direct identification of a single virus I: FASTER CARS.

From the famous 1918 H1N1 influenza to the present COVID-19 pandemic, the need for improved viral detection techniques is all too apparent. The aim of the present paper is to show that identification of individual virus particles in clinical sample materials quickly and reliably is near at hand. First of all, our team has developed techniques for identification of virions based on a modular atomic force microscopy (AFM). Furthermore, femtosecond adaptive spectroscopic techniques with enhanced resolution via coherent anti-Stokes Raman scattering (FASTER CARS) using tip-enhanced techniques markedly improves the sensitivity [M. O. Scully, et al, Proc. Natl. Acad. Sci. U.S.A. 99, 10994-11001 (2002)].

Novel reassortant swine H3N2 influenza A viruses in Germany.

Analysis of 228 H3N2 swine influenza A virus isolates collected between 2003 and 2015 in Germany revealed important changes in molecular epidemiology. The data indicate that a novel reassortant, Rietberg/2014-like swine H3N2, emerged in February 2014 in Northern Germany. It is comprised of a hemagglutinin gene of seasonal H3N2 (A/Denmark/129/2005-like), a neuraminidase gene of Emmelsbuell/2009-like swine H1N2 and the internal gene cassette of pandemic H1N1 viruses. Together with Danish swine H3N2 strains of 2013-2015 with identical genome layout, the Rietberg/2014-like viruses represent a second swine H3N2 lineage which cocirculates with a variant of the Gent/1984-like swine H3N2 lineage. This variant, named Gent1984/Diepholz-like swine H3N2, has a Gent/1984-like HA and a Diepholz/2008-like NA; the origin of the internal gene cassette likely derived from avian-like swine H1N1. The first isolate of the Gent1984/Diepholz reassortant emerged in Northern Germany in September 2011 whereas the last German Gent/1984-like isolate was collected in October 2011.

Cocirculation of Swine H1N1 Influenza A Virus Lineages in Germany.

The genome analysis of 328 H1N1 swine influenza virus isolates collected in a 13-year long-term swine influenza surveillance in Germany is reported. Viral genomes were sequenced with the Illumina next-generation sequencing technique and conventional Sanger methods. Phylogenetic analyses were conducted with Bayesian tree inference. The results indicate continued prevalence of Eurasian avian swine H1N1 but also emergence of a novel H1N1 reassortant, named Schneiderkrug/2013-like swine H1N1, with human-like hemagglutinin and avian-like neuraminidase and internal genes. Additionally, the evolution of an antigenic drift variant of A (H1N1) pdm09 was observed, named Wachtum/2014-like swine H1N1. Both variants were first isolated in northwest Germany, spread to neighboring German states and reached greater proportions of the H1N1 isolates of 2014 and 2015. The upsurge of Wachtum/2014-like swine H1N1 is of interest as this is the first documented persistent swine-to-swine spread of A (H1N1) pdm09 in Germany associated with antigenic variation. Present enzootic swine influenza viruses in Germany now include two or more co-circulating, antigenically variant viruses of each of the subtypes, H1N1 and H1N2.

Epidemiology of bacteria and viruses in the respiratory tract of humans and domestic pigs.

Bacteria and viruses were analysed in the upper respiratory tract of symptomatic pig farmers and their domestic pigs. Eighty six human nasal and 495 (50 pools) porcine snout swabs were collected in Schleswig-Holstein, Germany. Staphylococcus (S.) aureus (62.8%, 54/86), human rhino- and coronaviruses (HRV, 29.1%, 25/86; HCoV, 16.3%, 14/86) were frequently detected in humans, while Haemophilus parasuis (90.0%, 45/50), Mycoplasma hyorhinis (78.6%, 11/14), Enterovirus G (EV-G, 56.0%, 28/50) and S. aureus (36.0%, 18/50), respectively, were highly prevalent in pigs. The detection of S. aureus in human follow-up samples indicates a carrier status. The methicillin-resistant phenotype (MRSA) was identified in 33.3% (18/54) of nasal swabs and in one of 18 (5.6%) pooled snout swabs that were tested positive for S. aureus. Strains were indicative of the livestock-associated clonal complex CC398, with t011 being the most common staphylococcal protein A type. Enterobacterales and non-fermenters were frequently isolated from swabs. Their detection in follow-up samples suggests a carrier status. All were classified as being non-multiresistant. There was no example for cross-species transmission of viruses. In contrast, transmission of S. aureus through occupational contact to pigs seems possible. The study contributes to the 'One Health' approach.

Using a mouse-adapted A/HK/01/68 influenza virus to analyse the impact of NS1 evolution in codons 196 and 231 on viral replication and virulence.

Seasonal influenza viruses circulating between 1918 and 2009 harboured two prevalent genetic variations in the NS1 coding region. A glutamic acid (E)-to-lysine (K) exchange at position 196 was reported to diminish the capacity of NS1 to control interferon induction. Furthermore, alterations at position 231 determine a carboxy-terminal extension of seven amino acids from 230 to 237 residues. Sequence analyses of NS1 of the last 90 years suggest that variations at these two positions are functionally linked. To determine the impact of the two positions on viral replication in vivo, we used a mouse-adapted variant of A/Hong Kong/01/68 (maHK68) (H3N2). maHK68 encodes an NS1 of 237 amino acids with lysine at position 196. A panel of recombinant maHK68 viruses was generated encoding NS1 variants that differed at positions 196 and 231. Our analyses showed a clear effect of the K-196-to-E exchange on interferon induction and virus virulence. These effects were further modulated by the loss of the seven-amino-acid extension. We propose that the combination of NS1 E-196 with the short C-terminal variant conferred a fitness advantage that is reflected by increased virulence in vivo. Notably, this particular NS1 constellation was observed for the pandemic 1918 H1N1 virus.

Novel enteric viruses in fatal enteritis of grey squirrels.

Astro- and kobuviruses infect both humans and animals. Here, we report on the disease history, detection and genomic characterization of novel astro- and kobuviruses from fatal diarrhoea of two juvenile grey squirrels. The virus particles had enterovirus-like morphology and a diameter of 28-32 nm. Next-generation sequencing confirmed astro- and kobuviruses and sequence analysis revealed typical astrovirus and picornavirus genome organizations. The astrovirus ORF2 sequence clustered with a clade of unassigned astroviruses, with marmot and rodent mamastroviruses as closest relatives. For the kobuvirus, divergences greater than 49.4 % for P1 and 43.5 % in the non-structural proteins indicated a novel species. However, phylogenetic analysis of the 3D polymerase showed that it clustered with that of the newly classified ludopivirus A1, suggesting a previous recombination event in the evolution of the kobuvirus. Our data provide further insights into the diversity of astro- and kobuviruses and broaden the spectrum of viruses infecting grey squirrels.

Viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection.

Respiratory tract infections (RTI) can take a serious course under immunosuppression. Data on the impact of the underlying pathogens are still controversial. Samples from the upper (n = 322) and lower RT (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. Exclusion criteria were presence of relevant cultivable microorganisms, C-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. Samples were tested by PCR for the presence of herpesviruses (HSV-1/-2; VZV; CMV; HHV6; EBV), adenoviruses, bocaviruses, entero-/rhinoviruses (HRV), parechoviruses, coronaviruses, influenza viruses (IV), parainfluenza viruses as well as for pneumoviruses (HMPV and RSV), and atypical bacteria (Mycoplasma pneumoniae, M.p.; Chlamydia pneumoniae, C.p.). Viral/bacterial genome equivalents were detected in more than two-thirds of specimens. Under immunosuppression, herpesviruses (EBV 30.9%/14.6%, p < 0.001; CMV 19.6%/7.9%, p < 0.001; HSV-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. Immunocompromised adults tended to present a higher RSV prevalence (6.4%/2.4%, p = 0.078). Immunocompetent patients were more frequently tested positive for IV (15.0%/5.8%, p = 0.001) and M.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an M.p. epidemic in 2011. About 41.8% of samples were positive for a single pathogen, and among them EBV (19.9%) was most prevalent followed by HRV (18.2%) and IV (16.6%). HSV-2 and C.p. were not found. Marked seasonal effects were observed for HRV, IV, and RSV. Differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. The exact contribution of some herpesviruses to the development of RTI remains unclear.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.