key: cord-1016146-2dsesgof
authors: Zlateva, Kalina T.; Coenjaerts, Frank E. J.; Crusio, Kelly M.; Lammens, Christine; Leus, Frank; Viveen, Marco; Ieven, Margareta; Spaan, Willy J. M.; Claas, Eric C. J.; Gorbalenya, Alexander E.
title: No novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide CODEHOP-based primers
date: 2012-09-28
journal: Arch Virol
DOI: 10.1007/s00705-012-1487-4
sha: e36e46559cc272e329ebf3c0dd599c82c3e0283a
doc_id: 1016146
cord_uid: 2dsesgof

Novel viruses might be responsible for numerous disease cases with unknown etiology. In this study, we screened 1800 nasopharyngeal samples from adult outpatients with respiratory disease symptoms and healthy individuals. We employed a reverse transcription (RT)-PCR assay and CODEHOP-based primers (CT12-mCODEHOP) previously developed to recognize known and unknown corona- and toroviruses. The CT12-mCODEHOP assay detected 42.0 % (29/69) of samples positive for human coronaviruses (HCoV), including HCoV-229 (1/16), HCoV-NL63 (9/17), and HCoV-OC43 (19/36), and additionally HCoV-HKU1 (3), which was not targeted by the diagnostic real-time PCR assays. No other coronaviruses were identified in the analyzed samples.

Respiratory tract infections (RTIs) are the most common diseases in humans and are responsible for significant morbidity and mortality worldwide, especially in young children, the elderly, and immunocompromised individuals. During the past decade, a growing variety of new viruses has been implicated in human RTIs, including severe acute respiratory syndrome coronavirus (SARS-HCoV) [1] [2] [3] , human CoVs (HCoV) NL63 [4, 5] and HKU1 [6] , human metapneumovirus (hMPV) [7] , human bocavirus (HBoV) [8] , WU and KI polyomaviruses (WUPyV and KIPyV, respectively) [9, 10] , and viruses that belong to the previously unrecognized species Human rhinovirus C (HRV-C) [11, 12] . These discoveries were facilitated by advancements in diagnostic methods. Still, in recently conducted studies, no causative agent could be established in 20 to 45 % of respiratory disease cases [13] [14] [15] [16] . These figures indicate that both the improvement of the current diagnostic methods and the search for new respiratory viruses must be continued.

The main challenge to the identification of new viruses is their unknown characteristics. One of the popular approaches that have succeeded is the use of either degenerate or consensus primers that recognize highly conserved genome regions of already known viruses. These primers can detect new pathogens that are closely related to members of established species. For the identification of distantly related unknown viruses, the combined consensus and degenerate primer design strategy implemented in the consensus-degenerate hybrid oligonucleotide primer (CODEHOP) technique proved to be superior [17] [18] [19] [20] .

Also, other genetic-based techniques that are sequence independent have been developed and used in virus discovery. For instance, the VIDISCA method, based on recognition of restricted enzyme cleavage sites, was introduced [5] and advanced by employing non-rRNA hexamers for random priming, high-throughput genome sequencing [21] , and sample pooling using centrifugation [22] . It is highly sensitive and unbiased, but samples with an abundance of background RNA/DNA may not be suitable for VIDISCA, and its processivity remains relatively limited. Thus, CODEHOP-and VIDISCA-based techniques are complementary rather than an alternative for virus discovery at this stage of technology development.

We have recently described the development and validation of modified CODEHOP-based primers that were designed to recognize distantly related corona-and toroviruses of the family Coronaviridae (CT12-mCODEHOP) in a reverse transcription (RT)-PCR assay [23] . These primers detected four known circulating human coronaviruses and two bovine toroviruses directly from biological specimens. Additionally, in silico predictions indicated that the designed primers may recognize coronaviruses belonging to most recently discovered lineages. These results demonstrated that the CT12-mCODEHOP one-step RT-PCR assay can be a suitable tool for identification of novel members of the family Coronaviridae. In the current study, we present results of the first large-scale application of this method to a human respiratory specimen collection. We detected only the four currently known coronaviruses, providing further support for the prevalence of these respiratory coronaviruses in the human population.

A total of 1800 nasopharyngeal swabs collected from 10 primary-care networks (based in Antwerp, Gent, Rotenburg, Utrecht, Lodz, Barcelona, Mataro, Jonkoping, Cardiff and Southampton) in seven European countries (Belgium, Germany, the Netherlands, Poland, Spain, Sweden and the United Kingdom), as part of the EU Framework 6 supported GRACE (Genomics to Combat Resistance Against Antibiotics in Community-Acquired LRTI in Europe) Network were included in this study [21, 24] . The specimens were obtained from immunocompetent adult outpatients of C18 years of age with respiratory illness and asymptomatic matched controls over three consecutive winter seasons during the period 2007-2010. The inclusion criteria for symptomatic patients were acute cough with duration of up to and including 28 days or individuals in whom the general practitioner suspected the presence of acute lower respiratory tract infection. Control samples collected from the same practices were obtained from immunocompetent adults with no signs of acute respiratory illness of the same gender and age within 5 years of the patient's age (younger or older). One thousand sixty-four samples were collected from symptomatic adults at their first visit to the general practitioner (V1 samples), 484 were follow-up samples (V2 samples) obtained 4 weeks later, and 252 specimens were from subjects without respiratory disease symptoms. Total DNA/RNA nucleic acids were extracted using a NucliSENS EasyMAG (bioMerieux, Grenoble, France) and stored at -80°C until further use. The samples were screened by in-house monoplex or multiplex real-time RT-PCR assays for the following respiratory pathogens: HCoV-229E, -OC43, and -NL63, human rhinovirus (HRV) A/B/C, parainfluenza virus (PIV) 1-4, hMPV, adenovirus (HAdV), HBoV, respiratory syncytial virus (RSV), influenza (Inf) A/B viruses, WUPyV, and KIPyV. In addition, bacterial infections were detected as well, partly by conventional culture, but also by molecular methods, for Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Bordetella sp., Streptococcus pneumoniae and Haemophilus spp.

For real-time PCR detection of HCoV, viral RNA was reverse-transcribed into cDNA by using a MultiScribe reverse transcriptase kit and random hexamers (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's guidelines, followed by inactivation of reverse transcriptase for 5 min at 95°C. HCoV type-specific primers and probes were based on the conserved regions of the nucleocapsid gene (Table 1) . Their suitability for use in a multiplex assay was confirmed in a separate test that excluded that they cross-interact. Samples were assayed in a 50-ll reaction mixture containing 20 ll of cDNA, TaqMan universal PCR master mix (PE Applied Biosystems), primers, and fluorogenic probes labeled with the 5 0 reporter dye 6-carboxy-fluorescein (FAM) and the 3 0 quencher dye 6-carboxy-tetramethyl-rhodamine (TAM-RA). Amplification and detection were performed with the Taqman 7900HT system, essentially as described elsewhere [25] . The presence of possible inhibitors of the amplification reaction was controlled by using a murine encephalomyocarditis virus (RNA) or phocine herpes virus (DNA) as internal controls. Coronavirus identification was performed by re-testing of the multiplex real-time PCR positive samples with the separate type-specific (monoplex) primer and probe sets. Interquartile median (IQM) and interquartile range (IQR) estimates were obtained with SPSS v17.

For virus discovery, GRACE samples were analyzed blindly in a pool of two as reported previously [23] . In brief, 8 ll of pooled RNA/DNA extractions containing 4 ll of each sample were digested with DNase using a DNase I Amplification Grade Kit, Life Technologies, Breda, The Netherlands) in a 10-ll final reaction volume following the manufacturer's instructions. The CT12-mCODEHOP RT-PCR assay was performed using a QIAGEN OneStep RT-PCR Kit (QIAGEN Benelux B.V., Venlo, The Netherlands) in a 50-ll final volume containing 2 lM A2 forward (5'-GGGIWGGGATTACMCIaartgygaymg-3') and 4 lM B1 reverse (5'-CCCASAIGWTGTIccnccnggytt-3') primers and 10 ll of the RNA sample. Amplification was carried out in a Biorad MyCycler (BioRad, Veenendaal, The Netherlands) using a touch-down thermocycling protocol as described elsewhere [23] . PCR products were analyzed in 2 % TAE agarose gels stained with ethidium bromide. Amplification products of about the expected 200-bp size were confirmed by individual re-testing of the pooled samples using 8 ll of RNA/DNA extract. The origin of the bands was established by sequence analysis of PCR products.

Employing a multiplex real-time PCR assay designed to detect HCoV-OC43, -229E and -NL63, 69 out of 1800 nasopharyngeal specimens were found to be positive and were subsequently typed using species-specific monoplex assays ( Table 2 ). The viral load in specimens containing viruses of different HCoV species was comparable, corresponding to median Ct values ranging between 26.8 and 27.9. Parallel screening of all specimens was conducted using the CT12-mCODEHOP RT-PCR assay in the search for new members of the family Coronaviridae. The assay amplified a virus sequence in 1.8 % (32/1800) of the tested samples and in 42.0 % (29/69) of the samples found to be HCoV positive in the real-time PCR assays. One specimen was positive for HCoV-229E, 9 for HCoV-NL63, and 19 for HCoV-OC43; viral loads were relatively high and characterized by a median interquartile Ct value of 25.32 (interquartile range, 22.03-27.52). Additionally, the CT12-mCODEHOP RT-PCR assay detected three samples that were positive for HCoV-HUK1, which was not targeted by the real-time PCR assays, bringing the total number of coronavirus-positives to 72 out of 1800 samples (4 %). The majority of coronaviruses 5.6 % (59/1064) were identified in V1 samples obtained from symptomatic patients during their first visit to the general practitioner, 1.9 % (9/484) were detected in V2 follow-up samples, and 1.6 % (4/252) in asymptomatic controls.

The CT12-mCODEHOP RT-PCR assay showed the highest sensitivity towards HCoV-OC43, closely followed by HCoV-NL63, and then HCoV-229E. HCoV-OC43 infection was confirmed in 52.8 % (19/36) of the samples typed by real-time PCR, with viral loads corresponding to a Ct interquartile range of 22.44-27.56 (median 26.21). The detection rate for HCoV-NL63 was 53 % (9/17), with a higher median interquartile viral load than for HCoV-OC43, corresponding to Ct 23.6. Only one (6.7 %) HCoV-229E sample with a high virus load (Ct 21.14) was confirmed. The viral load in positive samples not detected by the CT12-mCODEHOP RT-PCR assay (false negatives) was relatively low (interquartile Ct value range, 27.75-34.51, median, 30.68). False-negative HCoV-229E and HCoV-NL63 samples had a lower median interquartile Ct value, 28.3 and 28.9, respectively, in comparison to 32.32 for HCoV-OC43, indicating higher viral loads and a lower sensitivity of the assay towards HCoV-229E and HCoV-NL63. These results are in accordance with our previous findings using quantified copy RNA transcripts and HCoVpositive respiratory specimens [23] .

Our goal was to search for unknown respiratory human coronaviruses that are distantly related to known coronaviruses and are prototypes of new genera that might be circulating in the human population. No new coronaviruses were discovered in this study. Although this observation is negative and thus belongs to those rarely reported, its documentation is important for unbiased knowledgebuilding [26] . This study was the first, to our knowledge, in which a large collection of samples was screened using a primer set designed to recognize both corona-and toroviruses rather than targeting (human) coronaviruses exclusively (e.g., see ref. [27] ). Nonspecific amplification products were not observed for any of the analyzed specimens, including numerous samples that were positive for other virus pathogens (data in preparation), indicating that the CT12-mCODEHOP RT-PCR assay is highly selective with respect to coronaviruses in nasopharyngeal samples. The lack of false positives contributed to a relatively high Table 1 Species-specific primers and probes used for coronavirus detection in multiplex and monoplex PCR assays processivity and low cost of the assay that allowed us to analyze a large number of samples. In contrast, the rate of false negatives was considerable (*60 %) and varied for different HCoVs but was close to that established in our pilot study in which this assay was introduced [23] . If the false-negative rate of detection, especially for HCoV-229E, had been lower, even at the expense of an increase in false positives, our confidence about the lack of new coronaviruses in the analyzed samples probably would have been higher. Unfortunately, increasing the primer degeneracy in our assay, which controls sensitivity, was not practical. It is already high due to an enormous evolutionary distance between coronaviruses and toroviruses, which these primers were designed to cover [23] . The primer design was done according to an approach that minimizes bias with respect to any particular subset of coronaviruses, e.g., HCoVs, and thus covers the genetic diversity evenly. The validity of these primers was confirmed in in silico analysis of coronaviruses that were not used in the design of the CT12-mCODEHOP primers [23] . Thus, the observed falsenegative rate, obviously high by virus diagnostic standards, is the current state of the art for this method. Because of the characteristics of this method, the negative conclusion about new respiratory human coronaviruses reported in this study is restricted to pathogens that are comparable in their prevalence and load to the known circulating respiratory coronaviruses. Furthermore, we acknowledge that this conclusion may also depend on a number of other variables, including, but not limited to, the time frame, population and geographic location surveyed. Even with these limitations, we believe that the results we have obtained are a step forward in characterizing the natural diversity of human coronaviruses and how we study it. Improving the method and expanding the application of this assay to larger datasets that also include stool and cerebrospinal fluid clinical specimens obtained from acute disease cases of unknown etiology would be worth exploring. This line of research could also benefit from application of virus-discovery techniques that involve deep next-generation sequencing (e.g., see ref. [28] ). 

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Acknowledgments We thank our GRACE project colleagues Herman Goossens, Anton M. van Loon and all involved in the GRACE Primary Care Network for the opportunity to conduct this study, and Lia van der Hoek, Igor Sidorov, Chris Lauber and Michael Rozanov for advice and discussions. This study was partially supported by an EU FP6 GRACE grant (LSHM-CT12-2005-518226).