Human Metapneumovirus: Discovery, Characterisation and Associated Disease

__Abstract__ Acute respiratory tract infections are responsible for considerable morbidity and mortality and costs attributable to acute respiratory tract illnesses (ARI) are an important burden on national health care budgets. A variety of viruses, bacteria and fungi are associated with ARI. This thesis only focuses on viral causes of ARI Diagnosis of respiratory tract infections is important for adequate treatment of patients and community surveillance studying the spread of respiratory viruses forms the basis for preventive strategies. Despite the use of a variety of diagnostic assays, 15-50% of the samples obtained from persons suffering from ARI do not yield positive diagnoses, which is at least in part due to limitations of diagnostic procedures. However, a proportion of ARI may be caused by still unknown pathogens, providing an additional explanation for the relatively high proportion of negative laboratory diagnoses. This thesis describes the identification and characterisation of a hitherto unknown virus as the causative agent of ARI, wh

ADAR1: "Editor-in-Chief" of Cytoplasmic Innate Immunity

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

Armed oncolytic viruses: A kick-start for anti-tumor immunity

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

Determinants of the efficacy of viro-immunotherapy: A review

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 s

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 i

Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients

During a 17-month period, we performed retrospective analyses of the prevalence of and clinical symptoms associated with human metapneumovirus (hMPV) infection, among patients in a university hospital in The Netherlands. All available nasal-aspirate, throat-swab, sputum, and bronchoalveolar-lavage samples (N=1515) were tested for hMPV RNA by reverse-transcriptase polymerase chain reaction. hMPV RNA was detected in 7% of samples from patients with respiratory tract illnesses (RTIs) and was the second-most-detected viral pathogen in these patients during the last 2 winter seasons. hMPV was detected primarily in very young children and in immunocompromised individuals. In young children, clinical symptoms associated with hMPV infection were similar to those associated with human respiratory syncytial virus (hRSV) infection, but dyspnea, feeding difficulties, and hypoxemia were reported more frequently in hRSV-infected children. Treatment with antibiotics and corticosteroids was reported more frequently in hMPV-infected children. From these data, we conclude that hMPV is an important pathogen associated with RTI

Antigenic and genetic variability of human metapneumoviruses

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

Cerebrospinal fluid findings in an adult with human metapneumovirus–associated encephalitis

__To the Editor:__ Acute encephalitis/encephalopathy associated with human metapneumovirus (HMPV) has been documented in children. Recently, Fok et al. described an encephalitis case in an adult but were unable to test cerebrospinal fluid (CSF) for HMPV. Following authors’ recommendations, we performed diagnostic testing on the CSF of an adult with HMPV-associated encephalitis. A previously healthy 61-year-old man came to our institution with headache and seizures 5 days after onset of an influenza-like illness. A lumbar puncture on admission revealed pleocytosis (36 cells/μL) and a mononuclear predominance of 98%. Results of magnetic resonance imaging and computed tomography of the head and chest radiography on admission were inconclusive. The patient was treated in the intensive care unit for possible viral and bacterial meningoencephalitis. Although results of routine CSF-workup for infectious causes were unremarkable, total CSF protein level was elevated at 1.39 g/L (reference range 0.2–0.4 g/L). A nasopharyngeal swab specimen was positive for HMPV (cycle threshold 28.6) using duplex reverse transcription PCR (r-gene; Biomérieux, Marcy l’Etoile, France). However, HMPV reverse transcription PCR results were negative in the concurrent CSF sample. Immunofluorescence assays demonstrated HMPV IgG (serum titer 1:8,192; CSF titers 1:64 and 1:32). Indices calculated using the formula (IgGCSF HMPV/IgGSerum HMPV)/(IgGCSF total/IgGSerum total) were lower than the cut-off value of 4, indicating absence of intrathecal IgG against HMPV (Table). As in the study by Fok et al., our case supports consideration of HMPV as a causative agent of acute encephalitis after respiratory tract infection in adults. We could not demonstrate direct or indirect evidence of HMPV CSF invasion as the cause for HMPV-associated encephalitis in an adult, in contrast to a case in a child in which detection of HMPV in CSF suggested a causative role in acute encephalitis. Our data may point toward the role of nonspecific inflammatory response as the main pathogenic factor in HMPV-related encephalitis in adults

Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection

AbstractIt 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

Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages

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