Ronald Dijkman

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

Human Bocavirus Can Be Cultured in Differentiated Human Airway Epithelial Cells

ABSTRACT In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.

Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential

ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections. A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses

The IFNL4 gene is a recently discovered type III interferon, which in a significant fraction of the human population harbours a frameshift mutation abolishing the IFNλ4 ORF. The expression of IFNλ4 is correlated with both poor spontaneous clearance of hepatitis C virus (HCV) and poor response to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL‐10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion of IFNλ4 is impaired compared to that of IFNλ3, and this impairment is not due to a weak signal peptide, which was previously believed. We found that IFNλ4 gets N‐linked glycosylated and that this glycosylation is required for secretion. Nevertheless, this glycosylation is not required for activity. Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection.

Mosaic structure of human coronavirus NL63, one thousand years of evolution.

Abstract Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be ∼3×10−4 substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.

Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

ABSTRACT Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.

The dominance of human coronavirus OC43 and NL63 infections in infants

Abstract Background It is unknown to what extent the human coronaviruses (HCoVs) OC43, HKU1, 229E and NL63 infect healthy children. Frequencies of infections are only known for hospitalized children. Objectives Comparing infection frequencies in children who have mild infections with frequencies in children needing hospital uptake will determine whether infection by one of the four HCoVs leads to more severe disease. In addition, the sequence of seroconversions can reveal whether infection by one HCoV protects from infection by other HCoVs. Study design Two distinct study groups were monitored: healthy children and children hospitalized due to respiratory infection. HCoV natural infection rates in healthy children were obtained by serology in 25 newborns (followed 0–20months). The frequencies of severe HCoVs infection was determined by real time RT-PCR among 1471 hospitalized infants (<2-years old) with acute respiratory tract disease. Results The majority of healthy children seroconverted for HCoV-OC43 (n =19) and HCoV-NL63 (n =17), less for HCoV-HKU1 (n =9) and HCoV-229E (n =5). Notably, HCoV-HKU1 seroconversion was absent after HCoV-OC43 infection. Also HCoV-229E infection was rarely observed after HCoV-NL63 infection (1 out of 5). In the hospital 207 (14%) out of 1471 children were HCoV positive. Again we observed most infection by HCoV-OC43 (n =85) and HCoV-NL63 (n =60), followed by HCoV-HKU1 (n =47) and HCoV-229E (n =15). Conclusions HCoV-NL63 and HCoV-OC43 infections occur frequently in early childhood, more often than HCoV-HKU1 or HCoV-229E infections. HCoV-OC43 and HCoV-NL63 may elicit immunity that protects from subsequent HCoV-HKU1 and HCoV-229E infection, respectively, which would explain why HCoV-OC43 and HCoV-NL63 are the most frequently infecting HCoVs. There are no indications that infection by one of the HCoVs is more pathogenic than others.

Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism.

ABSTRACT The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains.

Human Parechovirus Type 1, 3, 4, 5, and 6 Detection in Picornavirus Cultures

ABSTRACT Picornavirus cultures that could not be typed in neutralization assays were analyzed by VP1 reverse transcription-PCR (RT-PCR) and a virus discovery tool (VIDISCA). Human parechoviruses (HPeVs) were frequently identified, among which were the uncommon isolates HPeV-4, HPeV-5, and HPeV-6. The HPeV-5 isolate could be amplified only by VIDISCA and not by VP1 RT-PCR.

Inhibition of Human Coronavirus NL63 Infection at Early Stages of the Replication Cycle

ABSTRACT Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, β-d-N4-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 μg/ml, 2 μM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 μM, >200 nM, >200 nM, >100 μM, and 80 μM, respectively). These agents may be investigated further for the treatment of coronavirus infections.