Publications by authors named "Saskia L Smits"

68 Publications

Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak.

PLoS One 2020 20;15(2):e0229326. Epub 2020 Feb 20.

Animal and Plant Health Agency (APHA) - Weybridge, Addlestone, Surrey, United Kingdom.

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229326PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032710PMC
May 2020

Nontraumatic Myelopathy in Malawi: A Prospective Study in an Area with High HIV Prevalence.

Am J Trop Med Hyg 2020 02;102(2):451-457

Division of Infectious Diseases and Tropical Medicine, University Hospital, LMU Munich, Munich, Germany.

Nontraumatic myelopathy causes severe morbidity and is not uncommon in Africa. Clinically, patients often present with paraplegia, and extrinsic cord compression and transverse myelitis are most common causes. Data on exact pathogenesis are scanty because of limitations in diagnostic methods. In Queen Elizabeth Central Hospital, Blantyre, Malawi, we recorded consecutive patients presenting with nontraumatic paraplegia for maximally 6 months between January and July 2010 and from March to December 2011. The diagnostic workup included imaging and examining blood, stool, urine, sputum, and cerebrospinal fluid (CSF) samples for infection. After discharge, additional diagnostic tests, including screening for virus infections, borreliosis, syphilis, and schistosomiasis, were carried out in the Netherlands. The clinical diagnosis was, thus, revised in retrospect with a more accurate final differential diagnosis. Of 58 patients included, the mean age was 41 years (range, 12-83 years) and the median time between onset and presentation was 18 days (range, 0-121 days), and of 55 patients tested, 23 (42%) were HIV positive. Spinal tuberculosis ( = 24, 41%), tumors ( = 16, 28%), and transverse myelitis ( = 6, 10%) were most common; in six cases (10%), no diagnosis could be made. The additional tests yielded evidence for CSF infection with , , Epstein-Barr virus (EBV), HHV-6, HIV, as well as a novel cyclovirus. The diagnosis of the cause of paraplegia is complex and requires access to an magnetic resonance imaging (MRI) scan and other diagnostic (molecular) tools to demonstrate infection. The major challenge is to confirm the role of detected pathogens in the pathophysiology and to design an effective and affordable diagnostic approach.
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http://dx.doi.org/10.4269/ajtmh.19-0209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008330PMC
February 2020

Machine-learning based patient classification using Hepatitis B virus full-length genome quasispecies from Asian and European cohorts.

Sci Rep 2019 12 11;9(1):18892. Epub 2019 Dec 11.

Department of Viroscience, Erasmus Medical Center, Rotterdam, 's-Gravendijkwal 230, 3015 CE, Rotterdam, Netherlands.

Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating 'quasispecies' populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification.
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http://dx.doi.org/10.1038/s41598-019-55445-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906359PMC
December 2019

Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance.

PLoS One 2018 19;13(7):e0200849. Epub 2018 Jul 19.

Department of Viroscience, Erasmus MC, Rotterdam, Zuid- Holland, The Netherlands.

Influenza viruses can cause severe life threatening infections in high-risk patients, including young children, the elderly and patients with compromised immunity due to underlying medical conditions or immunosuppressive treatment. The impaired immunity of these patients causes prolonged virus infection and combined with antiviral treatment facilitates the emergence of viruses with resistance mutations. The diverse nature of their immune status makes them a challenging group to study the impact of influenza virus infection and the efficacy of antiviral therapy. Immunocompromised ferrets may represent a suitable animal model to assess influenza virus infection and antiviral treatment strategies in immunocompromised hosts. Here, ferrets were given a daily oral solution of mycophenolate mofetil, tacrolimus and prednisolone sodium phosphate to suppress their immune system. Groups of immunocompromised and immunocompetent ferrets were inoculated with an A/H3N2 influenza virus and were subsequently treated with Oseltamivir or left untreated. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the throat and nose specimens to study virus replication during the course of infection. All immunocompromised ferrets had prolonged presence of viral RNA and a higher total amount of virus shedding compared to the immunocompetent ferrets. Although Oseltamivir reduced the total amount of virus shedding from the nose and throat of treated ferrets, it also resulted in the emergence of the neuraminidase R292K resistance substitution in all these animals, as determined by mutation specific RT-PCR and next-generation sequencing. No additional mutations that could be associated with the emergence of the R292K resistance mutation were detected. The immunocompromised ferret model can be used to study A/H3N2 virus shedding and is a promising model to study new antiviral strategies and the emergence of antiviral resistance in immunocompromised hosts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200849PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053203PMC
January 2019

Virus discovery analyses on post-mortem brain tissue and cerebrospinal fluid of schizophrenia patients.

Schizophr Res 2018 07 23;197:605-606. Epub 2018 Feb 23.

Department of Psychiatry, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, USA. Electronic address:

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http://dx.doi.org/10.1016/j.schres.2018.02.012DOI Listing
July 2018

Transmission of Human Respiratory Syncytial Virus in the Immunocompromised Ferret Model.

Viruses 2018 01 2;10(1). Epub 2018 Jan 2.

Viroclinics Biosciences BV, Rotterdam 3029 AK, The Netherlands.

Human respiratory syncytial virus (HRSV) causes substantial morbidity and mortality in vulnerable patients, such as the very young, the elderly, and immunocompromised individuals of any age. Nosocomial transmission of HRSV remains a serious challenge in hospital settings, with intervention strategies largely limited to infection control measures, including isolation of cases, high standards of hand hygiene, cohort nursing, and use of personal protective equipment. No vaccines against HRSV are currently available, and treatment options are largely supportive care and expensive monoclonal antibody or antiviral therapy. The limitations of current animal models for HRSV infection impede the development of new preventive and therapeutic agents, and the assessment of their potential for limiting HRSV transmission, in particular in nosocomial settings. Here, we demonstrate the efficient transmission of HRSV from immunocompromised ferrets to both immunocompromised and immunocompetent contact ferrets, with pathological findings reproducing HRSV pathology in humans. The immunocompromised ferret-HRSV model represents a novel tool for the evaluation of intervention strategies against nosocomial transmission of HRSV.
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http://dx.doi.org/10.3390/v10010018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795431PMC
January 2018

Identification of HCV Resistant Variants against Direct Acting Antivirals in Plasma and Liver of Treatment Naïve Patients.

Sci Rep 2017 07 5;7(1):4688. Epub 2017 Jul 5.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma.
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http://dx.doi.org/10.1038/s41598-017-04931-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498547PMC
July 2017

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

Nature 2017 04 12;544(7650):309-315. Epub 2017 Apr 12.

Institute of Evolutionary Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3FL, UK.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.
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http://dx.doi.org/10.1038/nature22040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712493PMC
April 2017

Naturally occurring recombination in ferret coronaviruses revealed by complete genome characterization.

J Gen Virol 2016 09 7;97(9):2180-2186. Epub 2016 Jun 7.

Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.

Ferret coronaviruses (FRCoVs) exist as an enteric and a systemic pathotype, of which the latter is highly lethal to ferrets. To our knowledge, this study provides the first full genome sequence of a FRCoV, tentatively called FRCoV-NL-2010, which was detected in 2010 in ferrets in The Netherlands. Phylogenetic analysis showed that FRCoV-NL-2010 is most closely related to mink CoV, forming a separate clade of mustelid alphacoronavirus that split off early from other alphacoronaviruses. Based on sequence homology of the complete genome, we propose that these mustelid coronaviruses may be assigned to a new species. Comparison of FRCoV-NL-2010 with the partially sequenced ferret systemic coronavirus MSU-1 and ferret enteric coronavirus MSU-2 revealed that recombination in the spike, 3c and envelope genes occurred between different FRCoVs.
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http://dx.doi.org/10.1099/jgv.0.000520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079585PMC
September 2016

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe.

J Virol 2016 May 14;90(9):4269-4277. Epub 2016 Apr 14.

Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.

Unlabelled: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host.

Importance: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.
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http://dx.doi.org/10.1128/JVI.03046-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836327PMC
May 2016

An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels.

Science 2016 Jan 17;351(6268):77-81. Epub 2015 Dec 17.

Department of Viroscience, Erasmus Medical Center, Rotterdam, Netherlands. Artemis One Health, Utrecht, Netherlands. Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine, Hannover, Germany.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections have led to an ongoing outbreak in humans, which was fueled by multiple zoonotic MERS-CoV introductions from dromedary camels. In addition to the implementation of hygiene measures to limit further camel-to-human and human-to-human transmissions, vaccine-mediated reduction of MERS-CoV spread from the animal reservoir may be envisaged. Here we show that a modified vaccinia virus Ankara (MVA) vaccine expressing the MERS-CoV spike protein confers mucosal immunity in dromedary camels. Compared with results for control animals, we observed a significant reduction of excreted infectious virus and viral RNA transcripts in vaccinated animals upon MERS-CoV challenge. Protection correlated with the presence of serum neutralizing antibodies to MERS-CoV. Induction of MVA-specific antibodies that cross-neutralize camelpox virus would also provide protection against camelpox.
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http://dx.doi.org/10.1126/science.aad1283DOI Listing
January 2016

Molecular epidemiology and genetic diversity of hepatitis B virus in Ethiopia.

J Med Virol 2016 Jun 22;88(6):1035-43. Epub 2015 Dec 22.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

Although hepatitis B virus (HBV) infection is hyperendemic in Ethiopia and constitutes a major public health problem, little is known about its genetic diversity, genotypes, and circulation. The aim of this study was to determine the molecular epidemiology and genetic diversity of HBV in Ethiopia, using 391 serum samples collected from HBsAg-positive blood donors living in five different geographic regions. The HBV S/pol gene was amplified, sequenced, and HBV genotypes, subgenotypes, serotypes, and major hydrophilic region (MHR) variants were determined. Phylogenetic analysis of 371 samples (95%) revealed the distribution of genotypes A (78%) and D (22%) in Ethiopia. Further phylogenetic analysis identified one subgenotype (A1) within genotype A, and 4 subgenotypes within genotype D (D1; 1.3%, D2; 55%, D4; 2.5%, and D6; 8.8%). Importantly, 24 isolates (30%) of genotype D formed a novel phylogenetic cluster, distinct from any known D subgenotypes, and two A/D recombinants. Analysis of predicted amino-acid sequences within the HBsAg revealed four serotypes: adw2 (79%), ayw1 (3.1%), ayw2 (7.8%), and ayw3 (11.6%). Subsequent examination of sequences showed that 51 HBV isolates (14%) had mutations in the MHR and 8 isolates (2.2%) in the reverse transcriptase known to confer antiviral resistance. This study provides the first description of HBV genetic diversity in Ethiopia with a predominance of subgenotypes A1 and D2, and also identified HBV isolates that could represent a novel subgenotype. Furthermore, a significant prevalence of HBsAg variants in Ethiopian population is revealed.
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http://dx.doi.org/10.1002/jmv.24437DOI Listing
June 2016

Genotypic anomaly in Ebola virus strains circulating in Magazine Wharf area, Freetown, Sierra Leone, 2015.

Euro Surveill 2015 ;20(40)

Department of Viroscience, Erasmus Medical Center, Rotterdam, Netherlands.

The Magazine Wharf area, Freetown, Sierra Leone was a focus of ongoing Ebola virus transmission from late June 2015. Viral genomes linked to this area contain a series of 13 T to C substitutions in a 150 base pair intergenic region downstream of viral protein 40 open reading frame, similar to the Ebolavirus/H.sapiens-wt/SLE/2014/Makona-J0169 strain (J0169) detected in the same town in November 2014. This suggests that recently circulating viruses from Freetown descend from a J0169-like virus.
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http://dx.doi.org/10.2807/1560-7917.ES.2015.20.40.30035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946620PMC
April 2016

Recovering full-length viral genomes from metagenomes.

Front Microbiol 2015 1;6:1069. Epub 2015 Oct 1.

Department of Viroscience, Erasmus Medical Center Rotterdam, Netherlands.

Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.
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http://dx.doi.org/10.3389/fmicb.2015.01069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589665PMC
October 2015

Newly Identified Viruses in Human Gastroenteritis: Pathogens or Not?

Pediatr Infect Dis J 2016 Jan;35(1):104-7

From the *Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands; †Viroclinics Biosciences BV, Rotterdam, The Netherlands; ‡Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Hannover, Germany; §Artemis One Health, Utrecht, The Netherlands; and ¶Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

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http://dx.doi.org/10.1097/INF.0000000000000950DOI Listing
January 2016

High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.

Infect Ecol Epidemiol 2015 15;5:28305. Epub 2015 Jul 15.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

Two of the earliest Middle East respiratory syndrome (MERS) cases were men who had visited the Doha central animal market and adjoining slaughterhouse in Qatar. We show that a high proportion of camels presenting for slaughter in Qatar show evidence for nasal MERS-CoV shedding (62/105). Sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of MERS-CoV circulation and a high-risk area for human exposure. No correlation between RNA loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505336PMC
http://dx.doi.org/10.3402/iee.v5.28305DOI Listing
July 2015

Detection of Circovirus in Foxes with Meningoencephalitis, United Kingdom, 2009-2013.

Emerg Infect Dis 2015 Jul;21(7):1205-8

A fox circovirus was identified in serum samples from foxes with unexplained neurologic signs by using viral metagenomics. Fox circovirus nucleic acid was localized in histological lesions of the cerebrum by in situ hybridization. Viruses from the family Circoviridae may have neurologic tropism more commonly than previously anticipated.
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http://dx.doi.org/10.3201/eid2107.150228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480402PMC
July 2015

Asymptomatic Middle East respiratory syndrome coronavirus infection in rabbits.

J Virol 2015 Jun 25;89(11):6131-5. Epub 2015 Mar 25.

Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands Viroclinics Biosciences, Rotterdam, the Netherlands.

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.
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http://dx.doi.org/10.1128/JVI.00661-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442453PMC
June 2015

Reliable typing of MERS-CoV variants with a small genome fragment.

J Clin Virol 2015 Mar 15;64:83-7. Epub 2014 Dec 15.

Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands; Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven 3720BA, Netherlands. Electronic address:

Background: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples.

Objective: To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans.

Study Design: Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized.

Results: A reverse-transcription PCR assay for MERS-CoV targeting a 615 bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼ 35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9-32.2) yielded reliable sequence information in 14 samples.

Conclusions: We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location.
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http://dx.doi.org/10.1016/j.jcv.2014.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106551PMC
March 2015

Identification of protein receptors for coronaviruses by mass spectrometry.

Methods Mol Biol 2015 ;1282:165-82

Department of Viroscience, Erasmus Medical Center, 2040, Rotterdam, CA, 3000, The Netherlands.

As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.
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http://dx.doi.org/10.1007/978-1-4939-2438-7_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121102PMC
November 2015

Virus characterization and discovery in formalin-fixed paraffin-embedded tissues.

J Virol Methods 2015 Mar 12;214:54-9. Epub 2015 Feb 12.

Department of Viroscience, Erasmus MC, Dr. Molewaterplein 50, 3015GE Rotterdam, The Netherlands; Viroclinics Biosciences BV, Rotterdam, The Netherlands.

Detection and characterization of novel viruses is hampered frequently by the lack of properly stored materials. Especially for the retrospective identification of viruses responsible for past disease outbreaks, often only formalin-fixed paraffin-embedded (FFPE) tissue samples are available. Although FFPE tissues can be used to detect known viral sequences, the application of FFPE tissues for detection of novel viruses is currently unclear. In the present study it was shown that sequence-independent amplification in combination with next-generation sequencing can be used to detect sequences of known and unknown viruses, although with relatively low sensitivity. These findings indicate that this technique could be useful for detecting novel viral sequences in FFPE tissues collected from humans and animals with disease of unknown origin, when other samples are not available. In addition, application of this method to FFPE tissues allows to correlate with the presence of histopathological changes in the corresponding tissue sections.
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http://dx.doi.org/10.1016/j.jviromet.2015.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119673PMC
March 2015

Assembly of viral genomes from metagenomes.

Front Microbiol 2014 18;5:714. Epub 2014 Dec 18.

Department of Viroscience, Erasmus Medical Center Rotterdam, Netherlands.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.
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http://dx.doi.org/10.3389/fmicb.2014.00714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270193PMC
January 2015

Pathogenesis of Middle East respiratory syndrome coronavirus.

J Pathol 2015 Jan;235(2):175-84

Department of Viroscience, Erasmus Medical Centre, Rotterdam, The Netherlands.

Human coronaviruses (CoVs) mostly cause a common cold that is mild and self-limiting. Zoonotic transmission of CoVs such as the recently identified Middle East respiratory syndrome (MERS)-CoV and severe acute respiratory syndrome (SARS)-CoV, on the other hand, may be associated with severe lower respiratory tract infection. This article reviews the clinical and pathological data available on MERS and compares it to SARS. Most importantly, chest radiographs and imaging results of patients with MERS show features that resemble the findings of organizing pneumonia, different from the lesions in SARS patients, which show fibrocellular intra-alveolar organization with a bronchiolitis obliterans organizing pneumonia-like pattern. These findings are in line with differences in the induction of cytopathological changes, induction of host gene responses and sensitivity to the antiviral effect of interferons in vitro when comparing both MERS-CoV and SARS-CoV. The challenge will be to translate these findings into an integrated picture of MERS pathogenesis in humans and to develop intervention strategies that will eventually allow the effective control of this newly emerging infectious disease.
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http://dx.doi.org/10.1002/path.4458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167882PMC
January 2015

Metagenomic survey for viruses in Western Arctic caribou, Alaska, through iterative assembly of taxonomic units.

PLoS One 2014 20;9(8):e105227. Epub 2014 Aug 20.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands; Viroclinics Biosciences, Rotterdam, The Netherlands.

Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-)emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive inventory of viruses present in wildlife. We report a metagenomic viral survey of the Western Arctic herd of barren ground caribou (Rangifer tarandus granti) in Alaska, USA. The presence of mammalian viruses in eye and nose swabs of 39 free-ranging caribou was investigated by random amplification combined with a metagenomic analysis approach that applied exhaustive iterative assembly of sequencing results to define taxonomic units of each metagenome. Through homology search methods we identified the presence of several mammalian viruses, including different papillomaviruses, a novel parvovirus, polyomavirus, and a virus that potentially represents a member of a novel genus in the family Coronaviridae.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105227PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139337PMC
May 2015

Exploring the potential of next-generation sequencing in detection of respiratory viruses.

J Clin Microbiol 2014 Oct 6;52(10):3722-30. Epub 2014 Aug 6.

Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands Viroclinics Biosciences, Rotterdam, the Netherlands

Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.
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http://dx.doi.org/10.1128/JCM.01641-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187785PMC
October 2014

Isolation of MERS coronavirus from a dromedary camel, Qatar, 2014.

Emerg Infect Dis 2014 Aug;20(8):1339-42

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.
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http://dx.doi.org/10.3201/eid2008.140663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111206PMC
August 2014

Novel divergent nidovirus in a python with pneumonia.

J Gen Virol 2014 Nov 25;95(Pt 11):2480-2485. Epub 2014 Jul 25.

Artemis Research Institute for Wildlife Health, Utrecht, The Netherlands.

The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin-neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.
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http://dx.doi.org/10.1099/vir.0.068700-0DOI Listing
November 2014

New viruses in idiopathic human diarrhea cases, the Netherlands.

Emerg Infect Dis 2014 Jul;20(7):1218-22

Emerging viral infections can be identified by using a viral metagenomics approach for clinical human material. Diarrhea samples of patients with unexplained gastroenteritis from the Netherlands were analyzed by using viral metagenomics. Novel circular DNA viruses, bufaviruses, and genogroup III picobirnaviruses were identified. These data expand our knowledge of the human virome.
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http://dx.doi.org/10.3201/eid2007.140190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073879PMC
July 2014