cCMP and cUMP phosphodiesterases in viral infections.

In bacteria, cCMP and cUMP have a key role in defense against infection with bacterial viruses. Bacteriophages encode phosphodiesterases (PDEs; 'nucleases'; Apyc1), which cleave cCMP/cUMP, counteracting this defense. We propose that PDEs are of broader biological relevance, including cCMP/cUMP-cleaving PDEs of eukaryotic viruses, which may constitute new drug targets.

Phage Therapy in Germany-Update 2023.

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages.

Recombinant Reporter Phage rTUN1::nLuc Enables Rapid Detection and Real-Time Antibiotic Susceptibility Testing of Klebsiella pneumoniae K64 Strains.

The emergence of multi-drug-resistant Klebsiella pneumoniae (Kp) strains constitutes an enormous threat to global health as multi-drug resistance-associated treatment failure causes high mortality rates in nosocomial infections. Rapid pathogen detection and antibiotic resistance screening are therefore crucial for successful therapy and thus patient survival. Reporter phage-based diagnostics offer a way to speed up pathogen identification and resistance testing as integration of reporter genes into highly specific phages allows real-time detection of phage replication and thus living host cells. Kp-specific phages use the host's capsule, a major virulence factor of Kp, as a receptor for adsorption. To date, 80 different Kp capsule types (K-serotypes) have been described with predominant capsule types varying between different countries and continents. Therefore, reporter phages need to be customized according to the locally prevailing variants. Recently, we described the autographivirus vB_KpP_TUN1 (TUN1), which specifically infects Kp K64 strains, the most predominant capsule type at the military hospital in Tunis (MHT) that is also associated with high mortality rates. In this work, we developed the highly specific recombinant reporter phage rTUN1::nLuc, which produces nanoluciferase (nLuc) upon host infection and thus enables rapid detection of Kp K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1::nLuc allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 h.

Clinical and virological features of first human monkeypox cases in Germany.

BACKGROUND: Monkeypox is a zoonotic orthopoxvirus infection endemic in central and western Africa. In May 2022, human monkeypox infections including human-to-human transmission were reported in a multi-country outbreak in Europe and North America. CASE PRESENTATIONS: Here we present the first two cases of monkeypox infection in humans diagnosed in Germany. We present clinical and virological findings, including the detection of monkeypox virus DNA in blood and semen. The clinical presentation and medical history of our patients suggest close physical contact during sexual interactions as the route of infection. CONCLUSION: Monkeypox requires rapid diagnosis and prompt public health response. The disease should be considered in the current situation especially the differential diagnosis of vesicular or pustular rash, particularly in patients with frequent sexual contacts. Most importantly, it is essential to raise awareness among all health professionals for the rapid and correct recognition and diagnosis of this disease, which is probably still underreported in Europe (Adler et al. in Lancet Infect Dis https://doi.org/10.1016/s1473-3099(22)00228-6 , 2022).

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

CHIKV strains Brazil (wt) and Ross (lab-adapted) differ with regard to cell host range and antiviral sensitivity and show CPE in human glioblastoma cell lines U138 and U251.

Chikungunya virus (CHIKV), a (re)emerging arbovirus, is the causative agent of chikungunya fever. To date, no approved vaccine or specific antiviral therapy are available. CHIKV has repeatedly been responsible for serious economic and public health impacts in countries where CHIKV epidemics occurred. Antiviral tests in vitro are generally performed in Vero-B4 cells, a well characterised cell line derived from the kidney of an African green monkey. In this work we characterised a CHIKV patient isolate from Brazil (CHIKVBrazil) with regard to cell affinity, infectivity, propagation and cell damage and compared it with a high-passage lab strain (CHIKVRoss). Infecting various cell lines (Vero-B4, A549, Huh-7, DBTRG, U251, and U138) with both virus strains, we found distinct differences between the two viruses. CHIKVBrazil does not cause cytopathic effects (CPE) in the human hepatocarcinoma cell line Huh-7. Neither CHIKVBrazil nor CHIKVRoss caused CPE on A549 human lung epithelial cells. The human astrocyte derived glioblastoma cell lines U138 and U251 were found to be effective models for lytic infection with both virus strains and we discuss their predictive potential for neurogenic CHIKV disease. We also detected significant differences in antiviral efficacies regarding the two CHIKV strains. Generally, the antivirals ribavirin, hydroxychloroquine (HCQ) and T-1105 seem to work better against CHIKVBrazil in glioblastoma cells than in Vero-B4. Finally, full genome analyses of the CHIKV isolates were done in order to determine their lineage and possibly explain differences in tissue range and antiviral compound efficacies.

Emerging SARS-CoV-2 variant B.1.1.7 reduces neutralisation activity of antibodies against wild-type SARS-CoV-2.

Spike-specific antibodies contribute significantly to the neutralising activity against SARS-CoV-2 and are important for the therapeutic effect of convalescent plasma. B.1.1.7 is a recently emerged variant of SARS-CoV-2 that has several mutations in the gene encoding for the spike-protein. To assess the potential effect these mutations could have on the neutralising efficacy of antibodies, we evaluated 96 serum samples from convalescent plasma donors collected before the first occurrence of B.1.1.7 and tested their neutralising effect on wild-type SARS-CoV-2 and B.1.1.7. We found that B.1.1.7 is more resistant to neutralisation by convalescent plasma from patients infected with wild-type SARS-CoV-2 with an overall decrease in neutralising activity of 47.7%. Thus, the neutralising effect of convalescent plasma should be determined against the major circulating virus clades whenever possible to ensure the best possible therapeutic effect.

Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia.

BACKGROUND: Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. RESULTS: In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014-2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. CONCLUSIONS: K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

Cardiovascular Injury Due to SARS-CoV-2.

Purpose of Review: The world is currently facing the largest global health crisis since the early 1900s due to a novel coronavirus. While SARS-CoV-2 infection causes predictable symptoms in COVID-19 patients, including upper respiratory distress and fever, the heterogeneity of manifestations is surprising. This review focuses on direct and indirect causes of myocardial injury in COVID-19 patients and highlights current knowledge, treatment strategies, and outstanding questions in the field. Recent Findings: Data are emerging that highlight the extent of cardiovascular involvement in COVID-19 patients, including evidence that SARS-CoV-2 causes myocarditis and increases cardiac risk. The incidence of cardiac injury is much greater in patients with severe disease presentation and those in intensive care. Summary: During the past year, COVID-19 patient mortality rates have improved due to tailored pharmacological treatments and patient management strategies that address the unique presentation of symptoms, which will hopefully also reduce the incidence of cardiac injury.

Prophylactic strategies to control chikungunya virus infection.

Chikungunya virus (CHIKV) is a (re)emerging arbovirus and the causative agent of chikungunya fever. In recent years, CHIKV was responsible for a series of outbreaks, some of which had serious economic and public health impacts in the affected regions. So far, no CHIKV-specific antiviral therapy or vaccine has been approved. This review gives a brief summary on CHIKV epidemiology, spread, infection and diagnosis. It furthermore deals with the strategies against emerging diseases, drug development and the possibilities of testing antivirals against CHIKV in vitro and in vivo. With our review, we hope to provide the latest information on CHIKV, disease manifestation, as well as on the current state of CHIKV vaccine development and post-exposure therapy.

Novel Nucleoside Analogues as Effective Antiviral Agents for Zika Virus Infections.

Previously considered a neglected flavivirus, Zika virus has recently emerged as a public health concern due to its ability to spread rapidly and cause severe neurological disorders, such as microcephaly in newborn babies from infected mothers, and Guillain-Barré syndrome in adults. Despite extensive efforts towards the identification of effective therapies, specific antivirals are still not available. As part of ongoing medicinal chemistry studies to identify new antiviral agents, we screened against Zika virus replication in vitro in a targeted internal library of small-molecule agents, comprising both nucleoside and non-nucleoside agents. Among the compounds evaluated, novel aryloxyphosphoramidate prodrugs of the nucleosides 2'-C-methyl-adenosine, 2-CMA, and 7-deaza-2'C-methyl-adenosine, 7-DMA, were found to significantly inhibit the virus-induced cytopathic effect in multiple relevant cell lines. In addition, one of these prodrugs exhibits a synergistic antiviral effect against Zika virus when applied in combination with an indirect antiviral agent, a l-dideoxy bicyclic pyrimidine nucleoside analogue, which potently inhibits vaccinia and measles viruses in vitro by targeting a host pathway. Our findings provide a solid basis for further development of an antiviral therapy for Zika virus infections, possibly exploiting a dual approach combining two different agents, one targeting the viral polymerase (direct-acting antiviral), the second targeting a host-directed autophagy mechanism.

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Current and Promising Antivirals Against Chikungunya Virus.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever (CHIKF) and is categorized as a(n) (re)emerging arbovirus. CHIKV has repeatedly been responsible for outbreaks that caused serious economic and public health problems in the affected countries. To date, no vaccine or specific antiviral therapies are available. This review gives a summary on current antivirals that have been investigated as potential therapeutics against CHIKF. The mode of action as well as possible compound targets (viral and host targets) are being addressed. This review hopes to provide critical information on the in vitro efficacies of various compounds and might help researchers in their considerations for future experiments.

Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.

Correction: Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

[This corrects the article DOI: 10.1371/journal.pone.0179291.].

ELISA for Molluscum Contagiosum Virus.

Molluscum contagiosum virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self-limiting. In contrast to orthopoxviruses, MCV infections tend to take a subacute clinical course but may persist for up to 12 months. Current numbers for MCV seroprevalence in different geographical areas are based on a variety of historical serological methods from complement fixation assays to MCV ELISAs based on purified MCV virions and MC133 antigen expressed in a Semliki Forest Virus expression system. A standardized ELISA for the assessment of MCV seroprevalence would be useful to determine global MCV seroprevalence. The methods described show that polypeptides derived from MCV open reading frames MC084 (residues V123 to R230 and V33 to G117), mc133 (residues M1 to N370), and glutathione S-transferase (GST)-H3L (residues I142 to W251) expressed in E. coli RIL+ as GST fusion proteins can be used to assess antibody binding in a GST capture ELISA. We show how the ELISA can be used to screen a panel of patient sera previously characterized with the mc084 V123-R230 ELISA. © 2017 by John Wiley & Sons, Inc.

Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

BACKGROUND: There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. MATERIALS AND METHODS: PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. RESULTS: Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 µg mL-1) a value comparable to aciclovir (EC50 = 0.18 µg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 µg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. CONCLUSIONS: The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

In vitro permeation and biological activity of punicalagin and zinc (II) across skin and mucous membranes prone to Herpes simplex virus infection.

Coadministration of pomegranate rind extract (PRE) and zinc (II) ions has recently been reported as a potential new topical treatment for Herpes simplex virus (HSV) infections. In the current work we examined the in vitro topical delivery of punicalagin (major phytochemical of PRE) and zinc from hydrogels across epithelial membranes that can become infected with HSV. Porcine epidermal, buccal and vaginal mucous membranes were excised and mounted in Franz diffusion cells and dosed with a simple hydrogel containing PRE and zinc sulphate (ZnSO4). The permeation of punicalagin and zinc were determined by HPLC and ICPMS respectively; punicalagin was also determined in the basal layers by reverse tape stripping. Receptor phases from the epidermal membrane experiment were also used to challenge HSV-1 in Vero host cells, and ex vivo porcine skin was used to probe COX-2 modulation. Punicalagin and zinc permeated each of the three test membranes, with significantly greater amounts of both delivered across the epidermal membrane. The amounts of punicalagin permeating the buccal and vaginal membranes were similar, although the amount of zinc permeating the vaginal membrane was comparatively very large - it is known that zinc interacts with vaginal mucosa. The punicalagin recovered by reverse tape stripping of the epidermal, buccal and vaginal membranes gave 0.47±0.016, 0.45±0.052 and 0.51±0.048nMcm-2 respectively, and were statistically the same (p<0.05). A 2.5 log reduction was achieved against HSV-1 using diffusion cell receptor phase, and COX-2 expression was reduced by 64% in ex vivo skin after 6h. Overall, a hydrogel containing 1.25mgmL-1 PRE and 0.25M ZnSO4 was able to topically deliver both the major bioactive compound within PRE and Zn (II) across all membranes and into the site specific region of Herpes simplex vesicular clusters, while maintaining potentiated virucidal and anti-inflammatory properties. This novel therapeutic system therefore has potential for the topical treatment of HSV infections.

Extensively drug-resistant New Delhi metallo-ß-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012.

Carriage of the New Delhi metallo-ß-lactamase variant 1 (NDM-1) enables drug resistance to move between communities and hospitals. In Bangladesh, we found the blaNDM-1 gene in 62% of environmental waters and in fermentative and nonfermentative gram-negative bacteria. Escherichia coli sequence type (ST) 101 was most commonly found, reflecting a common global relationship between ST101 and NDM-1.