4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1.

IMPORTANCE: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.

Zoonotic Infection With Pigeon Paramyxovirus Type 1 Linked to Fatal Pneumonia.

The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Pigeon paramyxovirus type 1 from a fatal human case induces pneumonia in experimentally infected cynomolgus macaques (Macaca fascicularis).

Although avian paramyxovirus type 1 is known to cause mild transient conjunctivitis in human beings, there are two recent reports of fatal respiratory disease in immunocompromised human patients infected with the pigeon lineage of the virus (PPMV-1). In order to evaluate the potential of PPMV-1 to cause respiratory tract disease, we inoculated a PPMV-1 isolate (hPPMV-1/Netherlands/579/2003) from an immunocompromised human patient into three healthy cynomolgus macaques (Macaca fascicularis) and examined them by clinical, virological, and pathological assays. In all three macaques, PPMV-1 replication was restricted to the respiratory tract and caused pulmonary consolidation affecting up to 30% of the lung surface. Both alveolar and bronchiolar epithelial cells expressed viral antigen, which co-localized with areas of diffuse alveolar damage. The results of this study demonstrate that PPMV-1 is a primary respiratory pathogen in cynomolgus macaques, and support the conclusion that PPMV-1 may cause fatal respiratory disease in immunocompromised human patients.

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture.

Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼ 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 µM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.

Excessive production and extreme editing of human metapneumovirus defective interfering RNA is associated with type I IFN induction.

Type I IFN production is one of the hallmarks of host innate immune responses upon virus infection. Whilst most respiratory viruses carry IFN antagonists, reports on human metapneumovirus (HMPV) have been conflicting. Using deep sequencing, we have demonstrated that HMPV particles accumulate excessive amounts of defective interfering RNA (DIs) rapidly upon in vitro passage, and that these are associated with IFN induction. Importantly, the DIs were edited extensively; up to 70% of the original A and T residues had mutated to G or C, respectively. Such high editing rates of viral RNA have not, to our knowledge, been reported before. Bioinformatics and PCR assays indicated that adenosine deaminase acting on RNA (ADAR) was the most likely editing enzyme. HMPV thus has an unusually high propensity to generate DIs, which are edited at an unprecedented high frequency. The conflicting published data on HMPV IFN induction and antagonism are probably explained by DIs in virus stocks. The interaction of HMPV DIs with the RNA-editing machinery and IFN responses warrants further investigation.

Human metapneumovirus infection of organoid-derived human bronchial epithelium represents cell tropism and cytopathology as observed in in vivo models.

Human metapneumovirus (HMPV), a member of the Pneumoviridae family, causes upper and lower respiratory tract infections in humans. In vitro studies with HMPV have mostly been performed in monolayers of undifferentiated epithelial cells. In vivo studies in cynomolgus macaques and cotton rats have shown that ciliated epithelial cells are the main target of HMPV infection, but these observations cannot be studied in monolayer systems. Here, we established an organoid-derived bronchial culture model that allows physiologically relevant studies on HMPV. Inoculation with multiple prototype HMPV viruses and recent clinical virus isolates led to differences in replication among HMPV isolates. Prolific HMPV replication in this model caused damage to the ciliary layer, including cilia loss at advanced stages post-infection. These cytopathic effects correlated with those observed in previous in vivo studies with cynomolgus macaques. The assessment of the innate immune responses in three donors upon HMPV and RSV inoculation highlighted the importance of incorporating multiple donors to account for donor-dependent variation. In conclusion, these data indicate that the organoid-derived bronchial cell culture model resembles in vivo findings and is therefore a suitable and robust model for future HMPV studies. IMPORTANCE: Human metapneumovirus (HMPV) is one of the leading causative agents of respiratory disease in humans, with no treatment or vaccine available yet. The use of primary epithelial cultures that recapitulate the tissue morphology and biochemistry of the human airways could aid in defining more relevant targets to prevent HMPV infection. For this purpose, this study established the first primary organoid-derived bronchial culture model suitable for a broad range of HMPV isolates. These bronchial cultures were assessed for HMPV replication, cellular tropism, cytopathology, and innate immune responses, where the observations were linked to previous in vivo studies with HMPV. This study exposed an important gap in the HMPV field since extensively cell-passaged prototype HMPV B viruses did not replicate in the bronchial cultures, underpinning the need to use recently isolated viruses with a controlled passage history. These results were reproducible in three different donors, supporting this model to be suitable to study HMPV infection.

The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles.

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment.

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50-100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

Human Metapneumovirus: lessons learned over the first decade.

It has been 10 years since human metapneumovirus (HMPV) was identified as a causative agent of respiratory illness in humans. Since then, numerous studies have contributed to a substantial body of knowledge on many aspects of HMPV. This review summarizes our current knowledge on HMPV, HMPV disease pathogenesis, and disease intervention strategies and identifies a number of areas with key questions to be addressed in the future.

Viral sensing by epithelial cells involves PKR- and caspase-3-dependent generation of gasdermin E pores.

Viral sensing in myeloid cells involves inflammasome activation leading to gasdermin pore formation, cytokine release, and cell death. However, less is known about viral sensing in barrier epithelial cells, which are critical to the innate immune response to RNA viruses. Here, we show that poly(I:C), a mimic of viral dsRNA, is sensed by NLRP1 in human bronchial epithelial cells, leading to inflammasome-dependent gasdermin D (GSDMD) pore formation via caspase-1. DsRNA also stimulated a parallel sensing pathway via PKR which activated caspase-3 to cleave gasdermin E (GSDME) to form active pores. Influenza A virus (IAV) infection of cells caused GSDME activation, cytokine release, and cell death, in a PKR-dependent but NLRP1-independent manner, involving caspase-8 and caspase-3. Suppression of GSDMD and GSDME expression increased IAV replication. These data clarify mechanisms of gasdermin cleavage in response to viral sensing and reveal that gasdermin pore formation is intrinsically antiviral in human epithelial cells.

Emergence and Potential Extinction of Genetic Lineages of Human Metapneumovirus between 2005 and 2021.

Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion (n = 144) and attachment (n = 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification. IMPORTANCE Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.

Comparison between intratumoral and intravenously administered oncolytic virus therapy with Newcastle disease virus in a xenograft murine model for pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a poor clinical prognosis and is usually a metastatic disease. In the last decades, oncolytic viro-immunotherapy has shown a promise as treatment strategy with encouraging results for a variety of tumors. Newcastle Disease Virus (NDV) is an oncolytic virus which selectively infects and damages tumors either by directly killing tumor cells or by promoting an anti-tumor immune response. Several studies have demonstrated that NDV strains with a multi-basic cleavage site (MBCS) in the fusion protein (F) have increased anti-tumor efficacy upon intratumoral injection in murine tumor models. However, intravenous injections, in which the oncolytic virus spreads systemically, could be more beneficial to treat metastasized PDAC in addition to the primary tumor. In this study, we compared the oncolytic efficacy and safety of intratumoral and intravenous injections with NDV containing an MBCS in F (NDV F3aa) in an immune deficient murine xenograft (BxPC3) model for PDAC. In this model, both intratumoral and intravenous injections with NDV F3aa induced anti-tumor efficacy as measured at 10 days after the first injection. Upon intravenous injection virus was detected in some of the tumors, indicating the systemic spread of the virus. Upon both treatments, mice did not display weight loss or abnormalities and treated mice did not secrete virus to the environment. These data demonstrate that intravenous injections of NDV F3aa can be applicable to treat metastasized cancers in immune deficient hosts without inflicting adverse effects.

Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.

Fetal Transfer of Human Metapneumovirus-Neutralizing Antibodies Is Reduced From Mothers Living With HIV-1.

Transplacental hMPV-neutralizing antibody transfer was reduced from mothers living with HIV-1. However, a comparison of antibody titers at birth between hMPV hospitalization cases at <6 months and matched controls suggested that reduced maternal antibody might not be the primary cause of the previously reported elevated hMPV risk in HIV-1-exposed infants.

Whole genome sequencing of human metapneumoviruses from clinical specimens using MinION nanopore technology.

Human metapneumovirus (HMPV), a member of the Pneumoviridae family, is a causative agent of respiratory illness in young children, the elderly, and immunocompromised individuals. Globally, viruses belonging to two main genetic lineages circulate, A and B, which are further divided into four genetic sublineages (A1, A2, B1, B2). Classical genotyping of HMPV is based on the sequence of the fusion (F) and attachment (G) glycoprotein genes, which are under direct antibody-mediated immune pressure. Whole genome sequencing provides more information than sequencing of subgenomic fragments and is therefore a powerful tool for studying virus evolution and disease epidemiology and for identifying transmission events and nosocomial outbreaks. Here, we report a robust method to obtain whole genome sequences for HMPV using MinION Nanopore technology. This assay is able to generate HMPV whole genome sequences from clinical specimens with good coverage of the highly variable G gene and is equally sensitive for strains of all four genetic HMPV sublineages. This method can be used for studying HMPV genetics, epidemiology, and evolutionary dynamics.

Clinical impact of human metapneumovirus infections before and during the COVID-19 pandemic.

BACKGROUND: The first outbreak of coronavirus disease 2019 (COVID-19) occurred in March 2020 in Europe, which is normally the peak incidence period of human metapneumovirus (HMPV) infections, implying cocirculation and potentially causing competition between them. METHODS: We investigated differences in clinical characteristics and outcomes of HMPV infections in hospitalized patients before (January 2016-28 February, 2020) and HMPV and COVID-19 during part of the COVID-19 pandemic (28 February, 2020-1 April, 2020). RESULTS: A total of 239 HMPV patients and 303 COVID-19 patients were included. Incidence of HMPV peaked in March. Despite a 324% increase in HMPV testing during the COVID-19 outbreak, incidence of HMPV remained stable. Clinical characteristics showed 25 (11%) ICU admissions and 14 (6%) deaths. History of myocardial infarction, higher age and lower BMI were independently associated with increased 30-day mortality. Clinical characteristics of HMPV-infected patients did not differ between the non-COVID-19 period and the examined COVID-19 period except for length of hospital stay (7 vs. 5 days). HMPV infection and COVID-19 shared many clinical features but HMPV was associated with female gender, elderly patients and chronic conditions (COPD and chronic heart failure). Clinical outcomes did not differ between the viruses during the COVID-19 period. CONCLUSIONS: The clinical impact of HMPV infection did not change during the COVID-19 outbreak in terms of incidence and/or disease severity; hence, HMPV and SARS-CoV-2 are probably co-circulating independently. Despite the current clinical focus on the COVID-19 pandemic, clinicians should keep in mind that HMPV-infection may mimic COVID-19 and is also associated with serious adverse outcomes.

Rintatolimod Induces Antiviral Activities in Human Pancreatic Cancer Cells: Opening for an Anti-COVID-19 Opportunity in Cancer Patients?

Severe acute respiratory virus-2 (SARS-CoV-2) has spread globally leading to a devastating loss of life. Large registry studies have begun to shed light on the epidemiological and clinical vulnerabilities of cancer patients who succumb to or endure poor outcomes of SARS-CoV-2. Specific treatment for COVID-19 infections in cancer patients is lacking while the demand for treatment is increasing. Therefore, we explored the effect of Rintatolimod (Ampligen®) (AIM ImmunoTech, Ocala, FL, USA), a Toll-like receptor 3 (TLR3) agonist, to treat uninfected human pancreatic cancer cells (HPACs). The direct effect of Rintatolimod was measured by targeted gene expression profiling and by proteomics measurements. Our results show that Rintatolimod induces an antiviral effect in HPACs by inducing RNase-L-dependent and independent pathways of the innate immune system. Treatment with Rintatolimod activated the interferon signaling pathway, leading to the overexpression of several cytokines and chemokines in epithelial cells. Furthermore, Rintatolimod treatment increased the expression of angiogenesis-related genes without promoting fibrosis, which is the main cause of death in patients with COVID-19. We conclude that Rintatolimod could be considered an early additional treatment option for cancer patients who are infected with SARS-CoV-2 to prevent the complicated severity of the disease.

Developing oncolytic viruses for clinical use: A consortium approach.

The use of oncolytic viruses forms an appealing approach for cancer treatment. On the one hand the viruses replicate in, and kill, tumor cells, leading to their intra-tumoral amplification. On the other hand the viral infection will activate virus-directed immune responses, and may trigger immune responses directed against tumor cells and tumor antigens. To date, a wide variety of oncolytic viruses is being developed for use in cancer treatment. While the development of oncolytic viruses has often been initiated by researchers in academia and other public institutions, a large majority of the final product development and the testing of these products in clinical trials is industry led. As a consequence relatively few pre-clinical and clinical studies evaluated different oncolytic viruses in competitive side-by-side preclinical or clinical studies. In this review we will summarize the steps and considerations essential in the development and characterization of oncolytic viruses, and describe our multidisciplinary academic consortium, which involves a dozen departments in three different Dutch universities, collaborating in the development of oncolytic viruses. This consortium has the ambition to develop a small series of oncolytic viruses and to evaluate these in various cancers.

Immunometabolism pathways as the basis for innovative anti-viral strategies (INITIATE): A Marie Sklodowska-Curie innovative training network.

The past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human health. As illustrated by the global COVID19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks. Pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. While molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. A detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. INITIATE is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), with the goal to train 15 early stage PhD researchers (ESRs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). To this end, INITIATE brings together a highly complementary international team of academic and corporate leaders from 7 European countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. The ESRs of INITIATE are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. INITIATE will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases.