Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Detection of mpox virus in ambient air in a sexual health clinic.

Although transmitted mainly through direct (sexual) contact, mpox virus (MPXV) can be detected in ambient air. We explored the use of air sampling for diagnosis or (genomic) surveillance of mpox in a sexual health clinic. For six out of six patients who were infected with MPXV, all four of our ambient air PCR tests were positive. For 14 uninfected patients, PCR was positive in three ambient air samples, albeit with higher cycle threshold (Ct) values. Genomic sequencing of samples from two positive patients showed matching sequences between air and clinical samples.

Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks.

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes.

Indoor air surveillance and factors associated with respiratory pathogen detection in community settings in Belgium.

Currently, the real-life impact of indoor climate, human behaviour, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings in Belgium for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. Pathogen detection and concentration varied significantly by pathogen, month, and age group in generalised linear (mixed) models and generalised estimating equations. High CO2 and low natural ventilation were independent risk factors for detection. The odds ratio for detection was 1.09 (95% CI 1.03-1.15) per 100 parts per million (ppm) increase in CO2, and 0.88 (95% CI 0.80-0.97) per stepwise increase in natural ventilation (on a Likert scale). CO2 concentration and portable air filtration were independently associated with pathogen concentration. Each 100ppm increase in CO2 was associated with a qPCR Ct value decrease of 0.08 (95% CI -0.12 to -0.04), and portable air filtration with a 0.58 (95% CI 0.25-0.91) increase. The effects of occupancy, sampling duration, mask wearing, vocalisation, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.

Severity of COVID-19 among Hospitalized Patients: Omicron Remains a Severe Threat for Immunocompromised Hosts.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the general population in the context of a relatively high immunity gained through the early waves of coronavirus disease 19 (COVID-19), and vaccination campaigns. Despite this context, a significant number of patients were hospitalized, and identifying the risk factors associated with severe disease in the Omicron era is critical for targeting further preventive, and curative interventions. We retrospectively analyzed the individual medical records of 1501 SARS-CoV-2 positive hospitalized patients between 13 December 2021, and 13 February 2022, in Belgium, of which 187 (12.5%) were infected with Delta, and 1036 (69.0%) with Omicron. Unvaccinated adults showed an increased risk of moderate/severe/critical/fatal COVID-19 (crude OR 1.54; 95% CI 1.09-2.16) compared to vaccinated patients, whether infected with Omicron or Delta. In adults infected with Omicron and moderate/severe/critical/fatal COVID-19 (n = 323), immunocompromised patients showed an increased risk of in-hospital mortality related to COVID-19 (adjusted OR 2.42; 95% CI 1.39-4.22), compared to non-immunocompromised patients. The upcoming impact of the pandemic will be defined by evolving viral variants, and the immune system status of the population. The observations support that, in the context of an intrinsically less virulent variant, vaccination and underlying patient immunity remain the main drivers of severe disease.

Risk factors for disease severity and increased medical resource utilization in respiratory syncytial virus (+) hospitalized children: A descriptive study conducted in four Belgian hospitals.

BACKGROUND: We aimed to provide regional data on clinical symptoms, medical resource utilization (MRU), and risk factors for increased MRU in hospitalized respiratory syncytial virus (RSV)-infected Belgian pediatric population. METHODS: This prospective, multicenter study enrolled RSV (+) hospitalized children (aged ≤5y) during the 2013-2015 RSV seasons. RSV was diagnosed within 24h of hospitalization. Disease severity of RSV (+) patients was assessed until discharge or up to maximum six days using a Physical Examination Score (PES) and a derived score based on ability to feed, dyspnea and respiratory effort (PES3). MRU (concomitant medications, length of hospitalization [LOH], and oxygen supplementation) was evaluated. Kaplan-Meier survival analysis was performed to compare MRU by age and presence of risk factors for severe disease. Association between baseline covariates and MRU was analyzed using Cox regression models. RESULTS: In total, 75 children were included, Median (range) age was 4 (0-41) months, risk factors were present in 18.7%, and early hospitalization (≤3 days of symptom onset) was observed in 57.3% of patients. Cough (100%), feeding problems (82.2%), nasal discharge (87.8%), and rales and rhonchi (82.2%) were frequently observed. Median (range) LOH and oxygen supplementation was 5 (2-7) and 3 (1-7) days. Oxygen supplementation, bronchodilators, and antibiotics were administered to 58.7%, 64.0%, and 41.3% of the patients, respectively. Age <3 months and baseline total PES3 score were associated with probability and the duration of receiving oxygen supplementation. LOH was not associated with any covariate. CONCLUSION: RSV is associated with high disease burden and MRU in hospitalized children. Oxygen supplementation but not length of hospitalization was associated with very young age and the PES3 score. These results warrant further assessment of the PES3 score as a predictor for the probability of receiving and length of oxygen supplementation in RSV hospitalized children. REGISTRATION: NCT02133092.

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

INTRODUCTION: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. METHODS: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. RESULTS: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. CONCLUSION: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Proposal for Human Respiratory Syncytial Virus Nomenclature below the Species Level.

Human respiratory syncytial virus (HRSV) is the leading viral cause of serious pediatric respiratory disease, and lifelong reinfections are common. Its 2 major subgroups, A and B, exhibit some antigenic variability, enabling HRSV to circulate annually. Globally, research has increased the number of HRSV genomic sequences available. To ensure accurate molecular epidemiology analyses, we propose a uniform nomenclature for HRSV-positive samples and isolates, and HRSV sequences, namely: HRSV/subgroup identifier/geographic identifier/unique sequence identifier/year of sampling. We also propose a template for submitting associated metadata. Universal nomenclature would help researchers retrieve and analyze sequence data to better understand the evolution of this virus.

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

Towards a unified classification for human respiratory syncytial virus genotypes.

Since the first human respiratory syncytial virus (HRSV) genotype classification in 1998, inconsistent conclusions have been drawn regarding the criteria that define HRSV genotypes and their nomenclature, challenging data comparisons between research groups. In this study, we aim to unify the field of HRSV genotype classification by reviewing the different methods that have been used in the past to define HRSV genotypes and by proposing a new classification procedure, based on well-established phylogenetic methods. All available complete HRSV genomes (>12,000 bp) were downloaded from GenBank and divided into the two subgroups: HRSV-A and HRSV-B. From whole-genome alignments, the regions that correspond to the open reading frame of the glycoprotein G and the second hypervariable region (HVR2) of the ectodomain were extracted. In the resulting partial alignments, the phylogenetic signal within each fragment was assessed. Maximum likelihood phylogenetic trees were reconstructed using the complete genome alignments. Patristic distances were calculated between all pairs of tips in the phylogenetic tree and summarized as a density plot in order to determine a cutoff value at the lowest point following the major distance peak. Our data show that neither the HVR2 fragment nor the G gene contains sufficient phylogenetic signal to perform reliable phylogenetic reconstruction. Therefore, whole-genome alignments were used to determine HRSV genotypes. We define a genotype using the following criteria: a bootstrap support of ≥ 70 per cent for the respective clade and a maximum patristic distance between all members of the clade of ≤0.018 substitutions per site for HRSV-A or ≤0.026 substitutions per site for HRSV-B. By applying this definition, we distinguish twenty-three genotypes within subtype HRSV-A and six genotypes within subtype HRSV-B. Applying the genotype criteria on subsampled data sets confirmed the robustness of the method.

Comparison of the Idylla™ Respiratory (IFV-RSV) panel with the GeneXpert Xpert® Flu/RSV assay: a retrospective study with nasopharyngeal and midturbinate samples.

The objective of this study was to compare the performance of the Idylla™ Respiratory (IFV-RSV) panel to the GeneXpert Xpert® Flu/RSV assay and establish the performance of a midturbinate swab compared to nasopharyngeal sampling. Considering GeneXpert® assay as imperfect reference standard, a positive percentage agreement between both assays of 98-100% for influenza A and 96-99% for influenza B could be calculated when 354 nasopharyngeal and 325 midturbinate swabs were retrospectively analyzed. Comparing midturbinate samples to nasopharyngeal specimens of 321 subjects, positive percentage agreement varied from 42% to 94% depending on both target virus and assay used. Negative percentage agreements ranged from 98% to 100% for both methods and sample type comparison. The Idylla™ assay showed excellent performance compared to the GeneXpert® assay for the detection of influenza virus. The study also showed a slightly better performance for nasopharyngeal sampling compared to the use of a midturbinate swab.

Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection.

BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.

Prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in Belgium.

BACKGROUND: Acute Respiratory Infections (ARIs) are a major health problem, especially in young children and the elderly. OBJECTIVES: Insights into the seasonality of respiratory viruses can help us understand when the burden on society is highest and which age groups are most vulnerable. STUDY DESIGN: We monitored six respiratory viruses during five consecutive seasons (2011-2016) in Belgium. Patient specimens (n=22876), tested for one or more of the following respiratory viruses, were included in this analysis: Influenza viruses (IAV & IBV), Human respiratory syncytial virus (hRSV), Human metapneumovirus (hMPV), Adenovirus (ADV) and Human parainfluenza virus (hPIV). Data were analysed for four age categories: <6y, 6-17y, 18-64y and ≥65y. RESULTS: Children <6y had the highest infection rates (39% positive vs. 20% positive adults) and the highest frequency of co-infections. hRSV (28%) and IAV (32%) caused the most common respiratory viral infections and followed, like hMPV, a seasonal pattern with winter peaks. hRSV followed an annual pattern with two peaks: first in young children and ±7 weeks later in elderly. This phenomenon has not been described in literature so far. hPIV and ADV occurred throughout the year with higher rates in winter. CONCLUSIONS: Children <6y are most vulnerable for respiratory viral infections and have a higher risk for co-infections. hRSV and IAV are the most common respiratory infections with peaks during the winter season in Belgium.

In vivo evaluation of antiviral compounds on respiratory syncytial virus using a juvenile vervet monkey (Chlorocebus pygerythrus) infection model.

Several animal models with varying susceptibilities to respiratory syncytial virus (RSV) infection have been developed to study the specific aspects of RSV disease. Many of these models are used for testing antiviral compounds or in vaccine efficacy studies during preclinical evaluation. In this chapter, we describe the study design of an efficacy study of an RSV inhibitor, performed in a juvenile vervet monkey model for RSV.

Circulation of HRSV in Belgium: from multiple genotype circulation to prolonged circulation of predominant genotypes.

Molecular surveillance of HRSV in Belgium for 15 consecutive seasons (1996-2011) revealed a shift from a regular 3-yearly cyclic pattern, into a yearly alternating periodicity where HRSV-B is replaced by HRSV-A. Phylogenetic analysis for HRSV-A demonstrated the stable circulation of GA2 and GA5, with GA2 being dominant over GA5 during 5 consecutive seasons (2006-2011). We also identified 2 new genotype specific amino acid mutations of the GA2 genotype (A122 and Q156) and 7 new GA5 genotype specific amino acid mutations (F102, I108, T111, I125, D161, S191 and L217). Several amino acid positions, all located in the second hypervariable region of HRSV-A were found to be under positive selection. Phylogenetic analysis of HRSV-B showed the circulation of GB12 and GB13, where GB13 represented 100% of the isolated strains in 4 out of 5 consecutive seasons (2007-2011). Amino acids under positive selection were all located in the aminoterminal hypervariable region of HRSV-B, except one amino acid located in the conserved region. The genotype distribution within the HRSV-B subgroup has evolved from a co-circulation of multiple genotypes to the circulation of a single predominant genotype. The Belgian GB13 strains circulating since 2006, all clustered under the BAIV branch and contained several branch specific amino acid substitutions. The demographic history of genotypes GA2, GA5 and GB13 demonstrated a decrease in the total GA2 and GA5 population size, coinciding with the global expansion of the GB13 population. The emergence of the GB13 genotype resulted in a newly established balance between the predominant genotypes.

Susceptibility of the PER.C6 cell line for infection with clinical human respiratory syncytial virus isolates.

Human respiratory syncytial virus (HRSV) is one of the most important agents causing upper respiratory infection in infants. The susceptibility of the PER.C6 cell line for infection with clinical HRSV strains was investigated. During the HRSV season of 2006/2007 in Belgium, 90 isolates were inoculated into the PER.C6 and HEp-2 cells and viral growth was evaluated by quantification of the extracellular viral RNA concentration. Initially, viral growth of HRSV strains was observed after 7 days of inoculation in the PER.C6 cells, demonstrated by an increase in HRSV RNA levels. However, HRSV strains that were selected for continued propagation in the PER.C6 cells no longer seemed to replicate, which was reflected by consecutive decreasing viral RNA levels. Several approaches that were investigated to improve the efficiency of infection of clinical HRSV isolates in the PER.C6 cells, such as the influence of rotation and centrifugation of the virus inoculum into the cells were unsuccessful. In contrast, when clinical HRSV strains were inoculated in the HEp-2 cells substantial virus stocks could be produced and the virus could be propagated up to infectious titers of 5 logPFU per ml. In conclusion, the PER.C6 cell line was not permissive for persistent HRSV infection in our experiments and did not support long term production of large virus stocks at high titers. This possibly limits the use of this cell line for many in vitro applications using HRSV, e.g. plaque or virus yield reduction assays or the preparation of virus stocks for in vivo challenge experiments or in vitro vaccine production.

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses.

BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections.

Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice.

Until recently, human coronaviruses (HCoVs), such as HCoV strain OC43 (HCoV-OC43), were mainly known to cause 15 to 30% of mild upper respiratory tract infections. In recent years, the identification of new HCoVs, including severe acute respiratory syndrome coronavirus, revealed that HCoVs can be highly pathogenic and can cause more severe upper and lower respiratory tract infections, including bronchiolitis and pneumonia. To date, no specific antiviral drugs to prevent or treat HCoV infections are available. We demonstrate that chloroquine, a widely used drug with well-known antimalarial effects, inhibits HCoV-OC43 replication in HRT-18 cells, with a 50% effective concentration (+/- standard deviation) of 0.306 +/- 0.0091 microM and a 50% cytotoxic concentration (+/- standard deviation) of 419 +/- 192.5 microM, resulting in a selectivity index of 1,369. Further, we investigated whether chloroquine could prevent HCoV-OC43-induced death in newborn mice. Our results show that a lethal HCoV-OC43 infection in newborn C57BL/6 mice can be treated with chloroquine acquired transplacentally or via maternal milk. The highest survival rate (98.6%) of the pups was found when mother mice were treated daily with a concentration of 15 mg of chloroquine per kg of body weight. Survival rates declined in a dose-dependent manner, with 88% survival when treated with 5 mg/kg chloroquine and 13% survival when treated with 1 mg/kg chloroquine. Our results show that chloroquine can be highly effective against HCoV-OC43 infection in newborn mice and may be considered as a future drug against HCoVs.

Replication reduction neutralization test, a quantitative RT-PCR-based technique for the detection of neutralizing hantavirus antibodies.

Hantaviruses, which are mainly rodent-borne viruses, cause hemorrhagic fever with renal syndrome in the Old World, and hantavirus pulmonary syndrome in the New World. A neutralization test based on quantitative RT-PCR, the replication reduction neutralization test (RRNT), was developed for efficient detection of hantavirus-neutralizing antibodies. The effectiveness of the RRNT was evaluated by examining several hantaviruses and hantavirus-specific convalescent human serum samples. All convalescent serum samples tested by RRNT caused significant decreases in hantavirus genomes with only one specific hantavirus species, which allowed a straightforward identification of the related hantavirus. The results obtained by RRNT were completely comparable with the results obtained by focus reduction neutralization test (FRNT). The RRNT approach is a reliable and rapid alternative for FRNT, hitherto considered as the gold standard for hantavirus serology.

A pancoronavirus RT-PCR assay for detection of all known coronaviruses.

The recent discoveries of novel human coronaviruses, including the coronavirus causing SARS, and the previously unrecognized human coronaviruses HCoV-NL63 and HCoV-HKU1, indicate that the family Coronaviridae harbors more members than was previously assumed. All human coronaviruses characterized at present are associated with respiratory illnesses, ranging from mild common colds to more severe lower respiratory tract infections. Since the etiology of a relatively large percentage of respiratory tract diseases remains unidentified, it is possible that for a certain number of these illnesses, a yet unknown viral causative agent may be found. Screening for the presence of novel coronaviruses requires the use of a method that can detect all coronaviruses known at present. In this chapter, we describe a pancoronavirus degenerate primer-based method that allows the detection of all known and possibly unknown coronaviruses by RT-PCR amplification and sequencing of a 251-bp fragment of the coronavirus polymerase gene.