Publications by authors named "Marcel Jonges"

20 Publications

  • Page 1 of 1

Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples.

Antimicrob Resist Infect Control 2021 01 29;10(1):23. Epub 2021 Jan 29.

inBiome B.V., Science park 106, Amsterdam, The Netherlands.

Background: Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes.

Methods: We developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057-1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis.

Results: Ribosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed.

Conclusion: C. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.
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http://dx.doi.org/10.1186/s13756-020-00881-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845108PMC
January 2021

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

J Clin Virol 2021 Jan 18;134:104691. Epub 2020 Nov 18.

Department of Microbiology and Ecology, Faculty of Medicine, University of Valencia, Valencia, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jcv.2020.104691DOI Listing
January 2021

Follow-up of Contacts of Middle East Respiratory Syndrome Coronavirus-Infected Returning Travelers, the Netherlands, 2014.

Emerg Infect Dis 2015 Sep;21(9):1667-9

Notification of 2 imported cases of infection with Middle East respiratory syndrome coronavirus in the Netherlands triggered comprehensive monitoring of contacts. Observed low rates of virus transmission and the psychological effect of contact monitoring indicate that thoughtful assessment of close contacts is prudent and must be guided by clinical and epidemiologic risk factors.
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http://dx.doi.org/10.3201/eid2109.150560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550153PMC
September 2015

Wind-Mediated Spread of Low-Pathogenic Avian Influenza Virus into the Environment during Outbreaks at Commercial Poultry Farms.

PLoS One 2015 6;10(5):e0125401. Epub 2015 May 6.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands; Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.

Avian influenza virus-infected poultry can release a large amount of virus-contaminated droppings that serve as sources of infection for susceptible birds. Much research so far has focused on virus spread within flocks. However, as fecal material or manure is a major constituent of airborne poultry dust, virus-contaminated particulate matter from infected flocks may be dispersed into the environment. We collected samples of suspended particulate matter, or the inhalable dust fraction, inside, upwind and downwind of buildings holding poultry infected with low-pathogenic avian influenza virus, and tested them for the presence of endotoxins and influenza virus to characterize the potential impact of airborne influenza virus transmission during outbreaks at commercial poultry farms. Influenza viruses were detected by RT-PCR in filter-rinse fluids collected up to 60 meters downwind from the barns, but virus isolation did not yield any isolates. Viral loads in the air samples were low and beyond the limit of RT-PCR quantification except for one in-barn measurement showing a virus concentration of 8.48 x 10(4) genome copies/m(3). Air samples taken outside poultry barns had endotoxin concentrations of ~50 EU/m(3) that declined with increasing distance from the barn. Atmospheric dispersion modeling of particulate matter, using location-specific meteorological data for the sampling days, demonstrated a positive correlation between endotoxin measurements and modeled particulate matter concentrations, with an R(2) varying from 0.59 to 0.88. Our data suggest that areas at high risk for human or animal exposure to airborne influenza viruses can be modeled during an outbreak to allow directed interventions following targeted surveillance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125401PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422664PMC
April 2016

Improved detection of artifactual viral minority variants in high-throughput sequencing data.

Front Microbiol 2014 22;5:804. Epub 2015 Jan 22.

Department of Medical Microbiology, Academic Medical Centre Amsterdam, Netherlands.

High-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs).
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http://dx.doi.org/10.3389/fmicb.2014.00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302989PMC
February 2015

Travel-related MERS-CoV cases: an assessment of exposures and risk factors in a group of Dutch travellers returning from the Kingdom of Saudi Arabia, May 2014.

Emerg Themes Epidemiol 2014 17;11:16. Epub 2014 Oct 17.

National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Bilthoven, The Netherlands.

Background: In May 2014, Middle East respiratory syndrome coronavirus (MERS-CoV) infection, with closely related viral genomes, was diagnosed in two Dutch residents, returning from a pilgrimage to Medina and Mecca, Kingdom of Saudi Arabia (KSA). These patients travelled with a group of 29 other Dutch travellers. We conducted an epidemiological assessment of the travel group to identify likely source(s) of infection and presence of potential risk factors.

Methods: All travellers, including the two cases, completed a questionnaire focussing on potential human, animal and food exposures to MERS-CoV. The questionnaire was modified from the WHO MERS-CoV questionnaire, taking into account the specific route and activities of the travel group.

Results: Twelve non-cases drank unpasteurized camel milk and had contact with camels. Most travellers, including one of the two patients (Case 1), visited local markets, where six of them consumed fruits. Two travellers, including Case 1, were exposed to coughing patients when visiting a hospital in Medina. Four travellers, including Case 1, visited two hospitals in Mecca. All travellers had been in contact with Case 1 while he was sick, with initially non-respiratory complaints. The cases were found to be older than the other travellers and both had co-morbidities.

Conclusions: This epidemiological study revealed the complexity of MERS-CoV outbreak investigations with multiple potential exposures to MERS-CoV reported such as healthcare visits, camel exposure, and exposure to untreated food products. Exposure to MERS-CoV during a hospital visit is considered a likely source of infection for Case 1 but not for Case 2. For Case 2, the most likely source could not be determined. Exposure to MERS-CoV via direct contact with animals or dairy products seems unlikely for the two Dutch cases. Furthermore, exposure to a common but still unidentified source cannot be ruled out. More comprehensive research into sources of infection in the Arabian Peninsula is needed to strengthen and specify the prevention of MERS-CoV infections.
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http://dx.doi.org/10.1186/1742-7622-11-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200475PMC
October 2014

Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation.

Lancet Infect Dis 2014 Feb 17;14(2):140-5. Epub 2013 Dec 17.

Department of Viroscience, Erasmus Medical Center, Rotterdam, Netherlands; Centre for Infectious Disease Research, Diagnostics and Screening, Division of Virology, National Institute for Public Health and the Environment, Bilthoven, Netherlands. Electronic address:

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe lower respiratory tract infection in people. Previous studies suggested dromedary camels were a reservoir for this virus. We tested for the presence of MERS-CoV in dromedary camels from a farm in Qatar linked to two human cases of the infection in October, 2013.

Methods: We took nose swabs, rectal swabs, and blood samples from all camels on the Qatari farm. We tested swabs with RT-PCR, with amplification targeting the E gene (upE), nucleocapsid (N) gene, and open reading frame (ORF) 1a. PCR positive samples were tested by different MERS-CoV specific PCRs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. We tested serum samples from the camels for IgG immunofluorescence assay, protein microarray, and virus neutralisation assay.

Findings: We obtained samples from 14 camels on Oct 17, 2013. We detected MERS-CoV in nose swabs from three camels by three independent RT-PCRs and sequencing. The nucleotide sequence of an ORF1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the MERS-CoV from two human cases on the same farm and a MERS-CoV isolate from Hafr-Al-Batin. Eight additional camel nose swabs were positive on one or more RT-PCRs, but could not be confirmed by sequencing. All camels had MERS-CoV spike-binding antibodies that correlated well with the presence of neutralising antibodies to MERS-CoV.

Interpretation: Our study provides virological confirmation of MERS-CoV in camels and suggests a recent outbreak affecting both human beings and camels. We cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible.

Funding: European Union projects EMPERIE (contract number 223498), ANTIGONE (contract number 278976), and the VIRGO consortium.
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http://dx.doi.org/10.1016/S1473-3099(13)70690-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106553PMC
February 2014

Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A(H7N7) infection as determined by Illumina ultra-deep sequencing.

J Virol 2014 Feb 20;88(3):1694-702. Epub 2013 Nov 20.

Department of Virology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

Avian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.
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http://dx.doi.org/10.1128/JVI.02044-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911586PMC
February 2014

Genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza.

J Infect Dis 2013 Mar 10;207(5):730-5. Epub 2012 Dec 10.

Center for Infectious Disease Control, National Institute of Public Health and the Environment, Bilthoven, The Netherlands.

Outbreaks of highly pathogenic avian influenza in poultry can cause severe economic damage and represent a public health threat. Development of efficient containment measures requires an understanding of how these influenza viruses are transmitted between farms. However, the actual mechanisms of interfarm transmission are largely unknown. Dispersal of infectious material by wind has been suggested, but never demonstrated, as a possible cause of transmission between farms. Here we provide statistical evidence that the direction of spread of avian influenza A(H7N7) is correlated with the direction of wind at date of infection. Using detailed genetic and epidemiological data, we found the direction of spread by reconstructing the transmission tree for a large outbreak in the Netherlands in 2003. We conservatively estimate the contribution of a possible wind-mediated mechanism to the total amount of spread during this outbreak to be around 18%.
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http://dx.doi.org/10.1093/infdis/jis757DOI Listing
March 2013

Influenza A(H1N1) Oseltamivir Resistant Viruses in the Netherlands During the Winter 2007/2008.

Open Virol J 2011 23;5:154-62. Epub 2011 Dec 23.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Background: Antiviral susceptibility surveillance in the Netherlands was intensified after the first reports about the emergence of influenza A(H1N1) oseltamivir resistant viruses in Norway in January, 2008.

Methods: Within the existing influenza surveillance an additional questionnaire study was performed to retrospectively assess possible risk factors and establish clinical outcome of all patients with influenza virus A(H1N1) positive specimens. To discriminate resistant and sensitive viruses, fifty percent inhibitory concentrations for the neuramidase inhibitors oseltamivir and zanamivir were determined in a neuraminidase inhibition assay. Mutations previously associated with resistance to neuramidase inhibitors and M2 blockers (amantadine and rimantadine) were searched for by nucleotide sequencing of neuraminidase and M2 genes respectively.

Results: Among 171 patients infected with A(H1N1) viruses an overall prevalence of oseltamivir resistance of 27% (95% CI: 20-34%) was found. None of influenza A(H1N1) oseltamivir resistant viruses tested was resistant against amantadine or zanamivir. Patient characteristics, underlying conditions, influenza vaccination, symptoms, complications, and exposure to oseltamivir and other antivirals did not differ significantly between patients infected with resistant and sensitive A(H1N1) viruses.

Conclusion: In 2007/2008 a large proportion of influenza A(H1N1) viruses resistant to oseltamivir was detected. There were no clinical differences between patients infected with resistant and sensitive A(H1N1) viruses. Continuous monitoring of the antiviral drug sensitivity profile of influenza viruses is justified, preferably using the existing sentinel surveillance, however, complemented with data from the more severe end of the clinical spectrum. In order to act timely on emergencies of public health importance we suggest setting up a surveillance system that can guarantee rapid access to the latter.
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http://dx.doi.org/10.2174/1874357901105010154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249654PMC
August 2012

Impaired production of TNF-α by dendritic cells of older adults leads to a lower CD8+ T cell response against influenza.

Vaccine 2012 Feb 9;30(9):1659-66. Epub 2012 Jan 9.

Department of Vaccinology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

Seasonal influenza causes more morbidity and mortality in older adults than in young adults, apparently because of a decline in immune function with increasing age, known as immunosenescence. In this study, we compared the capacity of dendritic cells (DCs) from healthy older adults (≥65 years) with DCs from healthy young adults (20-40 years) to initiate a T cell response against influenza. DCs from older adults were impaired in the induction of influenza-specific CD8+ T cells as compared to DCs from young adults, which was demonstrated by a decreased proliferation, an impaired production of IFN-γ and a reduced expression of the degranulation marker CD107a by CD8+ T cells. Importantly, DCs from older adults produced significantly less TNF-α, showed a decreased expression of HLA class I and had a lower maturation state after influenza virus infection. Supplementing TNF-α increased the expression of HLA class I and of maturation markers and enhanced the induction of the influenza-specific CD8+ T cell response. Together, these findings indicate that the impaired influenza-specific CD8+ T cell response in older adults is associated with a reduced production of TNF-α and with a lower DC maturation. We suggest that the production of TNF-α is a determining factor in the DC-mediated CD8+ T cell response against influenza.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.105DOI Listing
February 2012

Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak.

J Virol 2011 Oct 17;85(20):10598-604. Epub 2011 Aug 17.

National Institute for Public Health and the Environment, Centre for Infectious Disease Control, P.O. Box 1, Bilthoven, Netherlands.

Although increasing data have become available that link human adaptation with specific molecular changes in nonhuman influenza viruses, the molecular changes of these viruses during a large highly pathogenic avian influenza virus (HPAI) outbreak in poultry along with avian-to-human transmission have never been documented. By comprehensive virologic analysis of combined veterinary and human samples obtained during a large HPAI A (H7N7) outbreak in the Netherlands in 2003, we mapped the acquisition of human adaptation markers to identify the public health risk associated with an HPAI outbreak in poultry. Full-length hemagglutinin (HA), neuraminidase (NA), and PB2 sequencing of A (H7N7) viruses obtained from 45 human cases showed amino acid variations at different codons in HA (n=20), NA (n=23), and PB2 (n=23). Identification of the avian sources of human virus infections based on 232 farm sequences demonstrated that for each gene about 50% of the variation was already present in poultry. Polygenic accumulation and farm-to-farm spread of known virulence and human adaptation markers in A (H7N7) virus-infected poultry occurred prior to farm-to-human transmission. These include the independent emergence of HA A143T mutants, accumulation of four NA mutations, and farm-to-farm spread of virus variants harboring mammalian host determinants D701N and S714I in PB2. This implies that HPAI viruses with pandemic potential can emerge directly from poultry. Since the public health risk of an avian influenza virus outbreak in poultry can rapidly change, we recommend virologic monitoring for human adaptation markers among poultry as well as among humans during the course of an outbreak in poultry.
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http://dx.doi.org/10.1128/JVI.05369-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187520PMC
October 2011

Oseltamivir-resistant pandemic A(H1N1) 2009 influenza viruses detected through enhanced surveillance in the Netherlands, 2009-2010.

Antiviral Res 2011 Oct 13;92(1):81-9. Epub 2011 Jul 13.

Center for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC50) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC50 was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure.
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http://dx.doi.org/10.1016/j.antiviral.2011.07.004DOI Listing
October 2011

Adamantane- and oseltamivir-resistant seasonal A (H1N1) and pandemic A (H1N1) 2009 influenza viruses in Guangdong, China, during 2008 and 2009.

J Clin Microbiol 2011 Jul 18;49(7):2651-5. Epub 2011 May 18.

Guangdong Center for Disease Control and Prevention, Guangzhou, Guangdong, People’s Republic of China.

Adamantane and oseltamivir resistance among influenza viruses is a major concern to public health officials. To determine the prevalence of antiviral-resistant influenza viruses in Guangdong, China, 244 seasonal A (H1N1) and 222 pandemic A (H1N1) 2009 viruses were screened for oseltamivir resistance by a fluorescence-based neuraminidase (NA) inhibition assay along with NA gene sequencing. Also, 147 seasonal A (H1N1) viruses were sequenced to detect adamantane resistance markers in M2. Adamantane-resistant seasonal A (H1N1) viruses clustering to clade 2C were dominant in 2008, followed by oseltamivir-resistant seasonal A (H1N1) viruses, clustering to clade 2B during January and May 2009. In June 2009, a lineage of double-resistant seasonal A (H1N1) viruses emerged, until it was replaced by the pandemic A (H1N1) 2009 viruses. The lineage most likely resulted from reassortment under the pressure of the overuse of adamantanes. As all viruses were resistant to at least one of the two types of antiviral agents, the need for close monitoring of the prevalence of antiviral resistance is stressed.
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http://dx.doi.org/10.1128/JCM.00535-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147846PMC
July 2011

Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding.

J Virol 2010 Nov 15;84(22):11802-13. Epub 2010 Sep 15.

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

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
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http://dx.doi.org/10.1128/JVI.01136-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977876PMC
November 2010

Introduction of virulence markers in PB2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission.

J Virol 2010 Apr 3;84(8):3752-8. Epub 2010 Feb 3.

Department of Virology, Erasmus Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.
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http://dx.doi.org/10.1128/JVI.02634-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849492PMC
April 2010

Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling.

J Clin Microbiol 2010 Mar 20;48(3):928-40. Epub 2010 Jan 20.

National Institute for Public Health and the Environment, Center for Infectious Disease Control, Laboratory for Infectious Diseases and Screening, Bilthoven, Netherlands.

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and beta-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and beta-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.
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http://dx.doi.org/10.1128/JCM.02045-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832438PMC
March 2010

Prolonged influenza virus infection during lymphocytopenia and frequent detection of drug-resistant viruses.

J Infect Dis 2009 May;199(10):1435-41

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

The factors that cause prolonged human influenza virus respiratory tract infection and determine its clinical impact and the development of drug-resistant viruses are unclear. During a 3-year period, symptomatic influenza virus excretion for 2 weeks was observed among 8 immunocompromised patients and found to be associated with lymphocytopenia at onset (8 of 8 patients) more often than with granulocytopenia (2 of 8 patients) or monocytopenia (2 of 8 patients). Six (75%) of 8 patients developed influenza lower respiratory tract infection (10 episodes), and receipt of oseltamivir treatment was significantly associated with clinical improvement (8 of 8 episodes vs. 0 of 2 untreated episodes; P = .02). Complete viral clearance was strongly correlated with lymphocyte reconstitution (P = .04) but was never observed during the first 2 weeks after oseltamivir treatment. Neuraminidase inhibitor-resistant influenza viruses emerged in 2 (67%) of 3 patients eligible for resistance analysis. In conclusion, prolonged influenza virus infection was associated with lymphocytopenia, influenza lower respiratory tract infection, and frequent development of drug resistance during antiviral therapy. Clinical improvement in influenza lower respiratory tract infection is observed during oseltamivir treatment, but complete viral clearance is dependent on lymphocyte reconstitution, irrespective of receipt of antiviral medication.
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http://dx.doi.org/10.1086/598684DOI Listing
May 2009

Morbidity and mortality associated with nosocomial transmission of oseltamivir-resistant influenza A(H1N1) virus.

JAMA 2009 Mar 2;301(10):1042-6. Epub 2009 Mar 2.

Department of Medical Microbiology, E4-65, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.

Context: The sudden emergence and rapid spread of oseltamivir-resistant influenza A(H1N1) viruses with neuraminidase (NA) gene H274Y amino acid substitution is the hallmark of global seasonal influenza since January 2008. Viruses carrying this mutation are widely presumed to exhibit attenuated pathogenicity, compromised transmission, and reduced lethality.

Objective: To investigate nosocomial viral transmission in a cluster of patients with influenza A(H1N1) virus infection.

Design, Setting, And Patients: Descriptive outbreak investigation of 2 hematopoietic stem cell transplant recipients and an elderly patient who developed hospital-acquired influenza A virus infection following exposure to an index patient with community-acquired H274Y-mutated influenza A(H1N1) virus infection in a medical ward at a Dutch university hospital in February 2008. The investigation included a review of the medical records, influenza virus polymerase chain reaction and culture, phenotypic oseltamivir and zanamivir susceptibility determination, and hemagglutinin chain 1 (HA(1)) gene and NA gene sequence analysis.

Main Outcome Measure: Phylogenetic relationship of patient cluster influenza A(H1N1) viruses and other 2007-2008 seasonal influenza A(H1N1) viruses.

Results: Viral HA(1) and NA gene sequence analysis from the 4 patients revealed indistinguishable nucleotide sequences and phylogenetic clustering of H274Y-mutated, oseltamivir-resistant influenza A(H1N1) virus, confirming nosocomial transmission. Influenza virus pneumonia (3 patients) and attributable mortality (2 patients) during active infection was observed in patients with lymphocytopenia at onset.

Conclusion: Seasonal oseltamivir-resistant influenza A(H1N1) viruses with NA gene H274Y mutation are transmitted and retain significant pathogenicity and lethality in high-risk patients.
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http://dx.doi.org/10.1001/jama.2009.297DOI Listing
March 2009

An amino acid substitution in the influenza A virus hemagglutinin associated with escape from recognition by human virus-specific CD4+ T-cells.

Virus Res 2007 Jun 8;126(1-2):282-7. Epub 2007 Apr 8.

Department of Virology and Postgraduate School Molecular Medicine, Erasmus Medical Center, Rotterdam, The Netherlands.

Influenza virus-specific CD4+ T-helper cells were cloned that recognized a virus strain isolated in 1981, but that failed to recognize more recent strains. The HLA-DR*1601-restricted epitope recognized was located in the hemagglutinin (HA(99-113)) and the naturally occurring A-->V substitution at position 106 was responsible for abrogating the recognition by HA(99-113)-specific CD4+ T-cells. This amino acid substitution was found in influenza A/H3N2 viruses that circulated between 1999 and 2005 and did not affect recognition by virus-specific antibodies. It was speculated that influenza viruses could evade recognition by virus-specific CD4+ T-cells, at least temporarily.
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http://dx.doi.org/10.1016/j.virusres.2007.02.018DOI Listing
June 2007