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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., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Y.Z. and M.R. are employees and hold stock in AbbVie.

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., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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.

The production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies. IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Newcastle Disease virus (NDV) has shown promise as an oncolytic virus for treatment of a wide range of tumours. NDV with a multi-basic cleavage site (MBCS) in the fusion (F) protein (NDV F3aa) has increased oncolytic efficacy in several tumour models, but also increased virulence in chickens compared to non-virulent NDV F0, raising potential environmental safety issues. Previously, we generated a variant of NDV F3aa with a disrupted V protein gene and a substitution of phenylalanine to serine at position 117 of the F protein (NDV F3aa-S- STOP V). Compared to NDV F3aa this virus had decreased virulence in embryonated chicken eggs. In this study, the virulence of the virus was evaluated upon inoculation of six-week-old chickens through a natural infection route and by determination of the intracerebral pathogenicity index (ICPI). Based on these data NDV F3aa-S- STOP V classified as a non-virulent virus. Although NDV F3aa was classified as a virulent virus based on the ICPI, the virus was also less pathogenic than NDV F0 upon inoculation of six-week-old chickens. These data indicate that NDV with a MBCS is not necessarily pathogenic in chickens. In addition, these data show that F3aa-S- STOP V is safe to use in viro-immunotherapies without posing a threat for chickens upon accidental exposure., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2021. Published by Elsevier B.V.)

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.

Oncolytic virus immunotherapy is rapidly gaining interest in the field of immunotherapy against cancer. The minimal toxicity upon treatment and the dual activity of direct oncolysis and immune activation make therapy with oncolytic viruses (OVs) an interesting treatment modality. The safety and efficacy of several OVs have been assessed in clinical trials and, so far, the Food and Drug Administration (FDA) has approved one OV. Unfortunately, most treatments with OVs have shown suboptimal responses in clinical trials, while they appeared more promising in preclinical studies, with tumours reducing after immune cell influx. In several clinical trials with OVs, parameters such as virus replication, virus-specific antibodies, systemic immune responses, immune cell influx into tumours and tumour-specific antibodies have been studied as predictors or correlates of therapy efficacy. In this review, these studies are summarized to improve our understanding of the determinants of the efficacy of OV therapies in humans and to provide insights for future developments in the viro-immunotherapy treatment field., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Specialized receptors that recognize molecular patterns such as double stranded RNA duplexes-indicative of viral replication-are potent triggers of the innate immune system. Although their activation is beneficial during viral infection, RNA transcribed from endogenous mobile genetic elements may also act as ligands potentially causing autoimmunity. Recent advances indicate that the adenosine deaminase ADAR1 through RNA editing is involved in dampening the canonical antiviral RIG-I-like receptor-, PKR-, and OAS-RNAse L pathways to prevent autoimmunity. However, this inhibitory effect must be overcome during viral infections. In this review we discuss ADAR1's critical role in balancing immune activation and self-tolerance.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae , including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1 , some of which are in the CIL domain. Using an Ifitm1 -/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization. IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo , and Ifitm1 -/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections., (Copyright © 2019 Smith et al.)

Oncolytic viruses (OVs), viruses that specifically result in killing tumor cells, represent a promising class of cancer therapy. Recently, the focus in the OV therapy field has shifted from their direct oncolytic effect to their immune stimulatory effect. OV therapy can function as a "kick start" for the antitumor immune response by releasing tumor associated antigens and release of inflammatory signals. Combining OVs with immune modulators could enhance the efficacy of both immune and OV therapies. Additionally, genetic engineering of OVs allows local expression of immune therapeutics, thereby reducing related toxicities. Different options to modify the tumor microenvironment in combination with OV therapy have been explored. The possibilities and obstacles of these combinations will be discussed in this review., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human metapneumovirus (HMPV), a single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae, is associated with respiratory tract illness, primarily in young children and persons with underlying disease. Based on genetic and antigenic variation, HMPV strains are classified into two serotypes, with isolates NL/1/00 and NL/1/99 as prototypes for serotypes A and B, respectively. The development of plasmid-based reverse genetics systems for both serotypes has resulted in developments of a wide range of vaccine candidates against HMPV infection. The approach to virus rescue of HMPV is similar to that used for other paramyxoviruses, starting with mini-replicon assays for optimizations of the rescue protocols and subsequent replacement of the mini genome with a plasmid expressing the cDNA of the full-length viral RNA genome. Here, we provide detailed information on the reverse genetics systems for HMPV.

The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Oncolytic viruses are a relatively new class of anti-cancer immunotherapy agents. Several viruses have undergone evaluation in clinical trials in the last decades, and the first agent is about to be approved to be used as a novel cancer therapy modality. In the current review, an overview is presented on recent (pre)clinical developments in the field of oncolytic viruses that have previously been or currently are being evaluated in clinical trials. Special attention is given to possible safety issues like toxicity, environmental shedding, mutation and reversion to wildtype virus.

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (© 2014 The Authors.)

Newcastle disease virus (NDV) is a naturally occurring oncolytic virus with clinically proven efficacy against several human tumor types. Selective replication in and killing of tumor cells by NDV is thought to occur because of differences in innate immune responses between normal and tumor cells. In our effort to develop oncolytic virotherapy with NDV for patients with pancreatic cancer, we evaluated the responses to NDV infection and interferon (IFN) treatment of 11 different established human pancreatic adenocarcinoma cell lines (HPACs). Here we show that all HPACs were susceptible to NDV. However, this NDV infection resulted in different replication kinetics and cytotoxic effects. Better replication resulted in more cytotoxicity. No correlation was observed between defects in the IFN pathways and NDV replication or NDV-induced cytotoxicity. IFN production by HPACs after NDV infection differed substantially. Pretreatment of HPACs with IFN resulted in diminished NDV replication and decreased the cytotoxic effects in most HPACs. These findings suggest that not all HPACs have functional defects in the innate immune pathways, possibly resulting in resistance to oncolytic virus treatment. These data support the rationale for designing recombinant oncolytic NDVs with optimized virulence that should likely contain an antagonist of the IFN pathways.

Paramyxoviruses, including measles virus (MV), human metapneumovirus (HMPV), human respiratory syncytial virus (HRSV) and canine distemper virus (CDV), are transmitted via the respiratory route. Despite their close phylogenetic relationship, the pathogenesis of these viruses is very different. To study viral tropism and replication ex vivo, a protocol for the inflation of lungs with low-melting-point agarose mixed with culture medium was established. Lung slices were prepared and remained viable in culture at 37°C for at least 7 days. Lung slices obtained from different animal species were infected with recombinant paramyxoviruses expressing enhanced green fluorescent protein (EGFP). Progression of infection was monitored in real time by detection of EGFP fluorescence. Flow cytometric analysis of cells emigrating into the culture medium proved to be a valuable tool for qualitative and quantitative assessment of infection over time. MV replicated optimally in lung slices of macaques, HMPV and HRSV in lung slices of cotton rats, and CDV in lung slices obtained from dogs and ferrets. In conclusion, these ex vivo lung slice cultures are discriminatory models for the study of respiratory virus infections and can be used to inform the design of future in vivo experiments, thereby reducing numbers of animals required., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract disease for which the development of vaccine candidates is warranted. We have previously described the generation of an iscom matrix-adjuvanted HMPV fusion protein subunit vaccine (Fsol) and a live-attenuated vaccine (HMPVM11). Here, we evaluate the immunogenicity and efficacy of these vaccines in cynomolgus macaques. Immunization with Fsol induced HMPV F-specific antibody responses, virus neutralizing antibody titers, and cellular immune responses, but the induced humoral immune response waned rapidly over time. HMPVM11 was strongly attenuated and displayed limited immunogenicity, although immunization with this virus primed for a good secondary HMPV-specific lymphoproliferative response after challenge infection. The duration of virus shedding in HMPVM11-immunized animals was reduced compared to sham-immunized animals. Both vaccines induced HMPV-specific immune responses, but the rapid waning of immunity is a challenging obstacle for vaccine development.

Human metapneumovirus (HMPV) causes acute respiratory tract illness primarily in young children, immunocompromised individuals and the elderly. Vaccines would be desirable to prevent severe illnesses in these risk groups. Here, we describe the generation and evaluation of cold-passage (cp) temperature-sensitive (ts) HMPV strains as vaccine candidates. Repeated passage of HMPV at low temperatures in Vero cells resulted in the accumulation of mutations in the viral genome. Introduction of these mutations in a recombinant HMPV by reverse genetics resulted in a ts-phenotype, judged on the decreased shut-off temperature for virus replication in vitro. As an alternative approach, three previously described cp-respiratory syncytial virus (cp-HRSV) mutations were introduced in a recombinant HMPV, which also resulted in a low shut-off temperature in vitro. Replication of these ts-viruses containing either the cp-HMPV or cp-HRSV mutations was reduced in the upper respiratory tract (URT) and undetectable in the lower respiratory tract (LRT) of hamsters. Nevertheless, high titres of HMPV-specific antibodies were induced by both ts-viruses. Upon immunization with the ts-viruses, the LRT of hamsters were completely protected against challenge infection with a heterologous HMPV strain, and URT viral titres were reduced by 10 000-fold. In conclusion, we provide proof-of-principle for two candidate live-attenuated HMPV vaccines that induce cross-protective immunity to prevent infection of the LRT in Syrian golden hamsters. Further mapping of the molecular determinants of attenuation of HMPV should be the subject of future studies.

Human metapneumovirus (HMPV) and avian metapneumovirus (AMPV) have a similar genome organization and protein composition, but a different host range. AMPV subgroup C (AMPV-C) is more closely related to HMPV than other AMPVs. To investigate the specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses, a minireplicon system was generated for AMPV-C and used in combination with minireplicon systems for HMPV lineages A1 and B1. Viral RNA-like molecules representing HMPV-A1 and -B1, AMPV-A and -C and human respiratory syncytial virus were replicated efficiently by polymerase complexes of HMPV-A1 and -B1 and AMPV-C, but not by polymerase complexes of bovine parainfluenza virus 3. Upon exchange of HMPV and AMPV-C polymerase complex components, all chimeric polymerase complexes were functional; exchange between HMPVs did not result in altered polymerase activity, whereas exchange between HMPVs and AMPV-C did. Recombinant HMPV-B1 viruses in which polymerase genes were exchanged with those of HMPV-A1 replicated with normal kinetics in vitro, whilst replacement with AMPV-C genes resulted in moderate differences in virus replication. In hamsters, recombinant HMPV-B1 viruses in which individual polymerase genes were exchanged with those of AMPV-C were attenuated, irrespective of the results obtained with minireplicon systems or in vitro replication assays. This study provides insight into the specificity and functional interaction of polymerase complex proteins of human and avian metapneumoviruses, but neither minireplicon systems nor in vitro replication kinetics were found to be predictive for attenuation in permissive animals.

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is an important cause of acute respiratory tract disease. In the 1960s, vaccination with formalin-inactivated paramyxovirus preparations--respiratory syncytial virus (RSV) and measles virus (MV)--resulted in predisposition for enhanced disease upon natural infection. We have produced a formalin-inactivated hMPV preparation (FI-hMPV), which was used to immunize young cynomolgus macaques. Six days after challenge FI-hMPV-primed monkeys had developed eosinophilic bronchitis and bronchiolitis, indicative of a hypersensitivity response. This study indicates that formalin-inactivated hMPV vaccines have the same propensity to predispose for immune-mediated disease as inactivated RSV and MV vaccines.

Human metapneumovirus (hMPV), a newly discovered paramyxovirus, is associated with acute respiratory-tract illness, primarily in young children, individuals with underlying disease and the elderly. Two genetic lineages of hMPV circulate around the world, and viruses from these two lineages demonstrate antigenic differences. The clinical impact of hMPV warrants the development of vaccines. Recombinant soluble fusion (F) proteins of prototype viruses of the two main lineages of hMPV that can be produced in high yields have been constructed. In this study, the antigenicity, immunogenicity and protective efficacy of these soluble F subunit vaccines were evaluated in Syrian golden hamsters (Mesocricetus auratus). Immunization of hamsters with the soluble F proteins, adjuvanted with Specol or iscom matrix, induced high virus-neutralization titres, with higher titres against the homologous than the heterologous virus. The neutralizing antibodies protected from subsequent infection of the lungs with both homologous and heterologous virus. Upon challenge, viral titres in the nasal turbinates of immunized animals were reduced significantly compared with those of PBS-immunized animals. In conclusion, a soluble F subunit vaccine for hMPV that induces cross-protective immunity for infection of the lower respiratory tract in Syrian golden hamsters has been generated.

Virus-neutralizing antibodies against human metapneumovirus (hMPV) have been shown to be important indicators for protection in experimental animal models. An improved plaque reduction virus neutralization assay to detect hMPV-specific neutralizing antibodies was designed using two prototype recombinant hMPV strains expressing green fluorescent protein (GFP). These prototypes represented each of the main antigenic variants of hMPV, because antigenic variability could have implications for vaccine development. The utility of mutations in the F gene resulting in trypsin-independent replication was also tested. Although these mutant hMPV strains could replicate in the absence of trypsin, bigger plaque size was achieved with the addition of trypsin. Insertion of the GFP gene in the genome of hMPV did not affect replication of the virus in vitro. Plaques could be detected by measuring expression of GFP after 5 days by automated scanning. Ferret, hamster, and macaque sera positive for hMPV were compared in a conventional virus neutralization assay and the plaque reduction virus neutralization assay. The results obtained with the two assays were in agreement but the improved plaque reduction virus neutralization assay was faster, more suitable for high throughput testing, and 10-fold more sensitive than the conventional virus neutralization assay.

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a causative agent of acute respiratory-tract illness. Two main hMPV lineages circulate worldwide and reinfections occur frequently. It is unclear what level of protection is induced by natural hMPV infection, what the durability of this protection is and whether it differs for reinfection with homologous or heterologous viruses. Here, protective immunity in cynomolgus macaques at different time points after inoculation with molecularly cloned prototype viruses of the two main lineages of hMPV has been addressed. Animals received a homologous challenge at 4, 6 or 12 weeks after the primary infection. In addition, animals that had been inoculated three times within 10 weeks were challenged with homologous or heterologous virus 8 months later. Primary infection with 10(7) TCID(50) resulted in virus shedding and induction of virus-neutralizing antibody responses, with higher titres against the homologous than the heterologous virus. Infections associated with virus shedding and seroconversion protected completely from homologous reinfection within 6 weeks, and partly at 12 weeks, after primary infection. Eight months later, protection had waned to virtually undetectable levels. This study demonstrates that experimental hMPV infection induces transient protective immunity.

Background: Human bocavirus is a newly discovered parvovirus. It has been detected primarily in children with acute lower respiratory tract infection, but its occurrence, clinical profile, and role as a causative agent of respiratory tract disease are not clear., Methods: We investigated the presence of human bocavirus by quantitative polymerase chain reaction of nasopharyngeal aspirate specimens and selected serum samples obtained from 259 children (median age, 1.6 years) who had been hospitalized for acute expiratory wheezing. The samples were analyzed for 16 respiratory viruses by polymerase chain reaction, virus culture, antigen detection, and serological assays., Results: At least 1 potential etiologic agent was detected in 95% of children, and >1 agent was detected in 34% of children. Human bocavirus was detected in 49 children (19%). A large proportion of the cases were mixed infections with other viruses, but human bocavirus was the only virus detected in 12 children (5%). High viral loads of human bocavirus were noted mainly in the absence of other viral agents, suggesting a causative role for acute wheezing. In addition, infections that had uncertain clinical relevance and low viral loads were prevalent. Human bocavirus DNA was frequently detected in serum specimens obtained from patients with acute wheezing, suggesting systemic infection., Conclusions: Human bocavirus is prevalent among children with acute wheezing and can cause systemic infection. Results suggest a model for bocavirus infection in which high viral loads are potentially associated with respiratory symptoms and low viral loads indicate asymptomatic shedding. Therefore, quantitative polymerase chain reaction analysis may be important for additional studies of human bocavirus.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

Human metapneumovirus (hMPV) was first described in Dutch children with acute respiratory symptoms. A prospective analysis of the epidemiology, clinical manifestation, and seroprevalence of hMPV and other respiratory viruses in South African children referred to hospital for upper or lower respiratory tract infection were carried out during a single winter season, by using RT-PCR, viral culture, and enzyme-linked immunosorbent assays. In nasopharyngeal aspirates from 137 children, hMPV was detected by RT-PCR in 8 (5.8%) children (2-43 months of age) as a sole viral pathogen, respiratory syncytial virus (RSV) in 21 (15%), influenza A virus in 18 (13%) and influenza B virus in 20 (15%). Pneumonia was diagnosed in seven children and upper respiratory tract infection in one of the hMPV-infected children. One hMPV-infected child was admitted to the intensive care unit in need of mechanical ventilation and one child was infected with human immunodeficiency virus (HIV). No statistically significant differences were found between hMPV, RSV, and influenza virus infected groups with regard to clinical signs and symptoms and chest radiograph findings. The seropositive rate of hMPV specific IgG antibodies was 92% in children aged 24-36 months, the oldest seronegative child in our study was 7 years and 6 months of age. In conclusion, hMPV contributes to upper and lower respiratory tract morbidity in South African children., (Copyright 2004 Wiley-Liss, Inc.)

A substantial proportion of hitherto unexplained respiratory tract illnesses is associated with human metapneumovirus (hMPV) infection. This virus also was found in patients with severe acute respiratory syndrome (SARS). To determine the dynamics and associated lesions of hMPV infection, six cynomolgus macaques (Macaca fascicularis) were inoculated with hMPV and examined by pathological and virological assays. They were euthanized at 5 (n = 2) or 9 (n = 2) days post-infection (dpi), or monitored until 14 dpi (n = 2). Viral excretion peaked at 4 dpi and decreased to zero by 10 dpi. Viral replication was restricted to the respiratory tract and associated with minimal to mild, multi-focal erosive and inflammatory changes in conducting airways, and increased numbers of macrophages in alveoli. Viral expression was seen mainly at the apical surface of ciliated epithelial cells throughout the respiratory tract, and less frequently in type 1 pneumocytes and alveolar macrophages. Both cell tropism and respiratory lesions were distinct from those of SARS-associated coronavirus infection, excluding hMPV as the primary cause of SARS. This study demonstrates that hMPV is a respiratory pathogen and indicates that viral replication is short-lived, polarized to the apical surface, and occurs primarily in ciliated respiratory epithelial cells.

Human metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae within the family Paramyxo- viridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups on the basis of genetic or antigenic differences or both. For HMPV, the existence of different genetic lineages has been described on the basis of variation in a limited set of available sequences. We address the antigenic relationship between genetic lineages in virus neutralization assays. In addition, we analyzed the genetic diversity of HMPV by phylogenetic analysis of sequences obtained for part of the fusion protein (n = 84) and the complete attachment protein open reading frames (n = 35). On the basis of sequence diversity between attachment protein genes and the differences in virus neutralization titers, two HMPV serotypes were defined. Each serotype could be divided into two genetic lineages, but these did not reflect major antigenic differences.

The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.

Background: Acute respiratory infections are the most common illnesses experienced by people of all ages worldwide. A portion of hitherto unexplained viral respiratory tract illnesses (RTIs) can now be attributed to the human metapneumovirus (hMPV), which was discovered in 2001. Several surveys on the burden of disease of hMPV infection have been conducted in various study groups and with different diagnostic assays. To estimate the impact of hMPV infection in a hospital setting and in the community at large, we reviewed these surveys to establish the burden of disease of hMPV infection., Methods: Published data and our own additional unpublished data on the clinical impact of hMPV infection were reviewed., Results and Conclusion: Worldwide, hMPV infections account for at least 5 to 7% of the RTI in hospitalized children, but immunocompromised and elderly individuals are also at risk. In the general community hMPV infections account for at least 3% of patients who visit a general practitioner for RTI. The seasonality of hMPV infections resembles that for respiratory syncytial virus and influenza virus infections, with recurrent epidemics during the winter months. Clinical symptoms and laboratory findings associated with hMPV infection exhibit a spectrum virtually indistinguishable from those associated with respiratory syncytial virus disease. The development of diagnostic assays must take into account the existence of two hMPV serotypes.