Saskia L. Smits

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.

Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study

Summary Background A new betacoronavirus—Middle East respiratory syndrome coronavirus (MERS-CoV)—has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. Methods We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. Findings 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. Interpretation MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. Funding European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft.

Human Coronavirus EMC Does Not Require the SARS-Coronavirus Receptor and Maintains Broad Replicative Capability in Mammalian Cell Lines

ABSTRACT A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is highly critical, because the SARS-CoV receptor influenced pathology, and its localization in the deep respiratory tract is thought to have restricted the transmissibility of SARS. Our data show that hCoV-EMC does not need the SARS-CoV receptor to infect human cells. Moreover, the virus is capable of infecting human, pig, and bat cells. This is remarkable, as human CoVs normally cannot replicate in bat cells as a consequence of host adaptation. Our results implicate that the new virus might use a receptor that is conserved between bats, pigs and humans suggesting a low barrier against cross-host transmission. A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is highly critical, because the SARS-CoV receptor influenced pathology, and its localization in the deep respiratory tract is thought to have restricted the transmissibility of SARS. Our data show that hCoV-EMC does not need the SARS-CoV receptor to infect human cells. Moreover, the virus is capable of infecting human, pig, and bat cells. This is remarkable, as human CoVs normally cannot replicate in bat cells as a consequence of host adaptation. Our results implicate that the new virus might use a receptor that is conserved between bats, pigs and humans suggesting a low barrier against cross-host transmission.

Identification of a Naturally Occurring Recombinant Genotype 2/6 Hepatitis C Virus

ABSTRACT Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently classified into six genotypes and an increasing number of subtypes. Most likely, this heterogeneity is caused by genetic drift; evidence for recombination is scarce. To study the molecular heterogeneity of HCV in Vietnam, we analyzed 58 HCV RNA-positive sera from Vietnamese blood donors by sequence analysis of the CORE and NS5B regions. Phylogenetic analyses revealed the presence of genotype 1 (38%), genotype 2 (10.3%), and genotype 6 viruses (51.7%). All samples showed concordant results except for two (D3 and D54). Sample D54 was a mixed infection of genotype 2i and 6h viruses. Whole-genome analysis and bootscan analysis of sample D3, on the other hand, revealed a recombinant virus with genotype 2i and genotype 6p sequences at the 5′ and 3′ ends, respectively. The crossover point was located between nucleotide positions 3405 to 3464 (numbering according to prototype strain HCV-H, M67463) at the NS2/NS3 junction. The identification of this naturally occurring recombinant virus strengthens the concept that recombination may play a role in HCV epidemiology and evolution. Furthermore, the location of the recombination breakpoint may be relevant for constructing infectious chimeric viruses.

Metagenomic analysis of the viral flora of pine marten and European badger feces

ABSTRACT A thorough understanding of the diversity of viruses in wildlife provides epidemiological baseline information about potential pathogens. Metagenomic analysis of the enteric viral flora revealed a new anellovirus and bocavirus species in pine martens and a new circovirus-like virus and geminivirus-related DNA virus in European badgers. In addition, sequences with homology to viruses from the families Paramyxo- and Picornaviridae were detected.

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

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.

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

Coronaviruses in the Middle East Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness and kills about a third of people infected. The virus is common in dromedary camels, which can be a source of human infections. In a survey for MERSCoV in over 1300 Saudi Arabian camels, Sabir et al. found that dromedaries share three coronavirus species with humans. Diverse MERS lineages in camels have caused human infections, which suggests that transfer among host species occurs quite easily. Haagmans et al. made a MERS-CoV vaccine for use in camels, using poxvirus as a vehicle. The vaccine significantly reduced virus excretion, which should help reduce the potential for transmission to humans, and conferred cross-immunity to camelpox infections. Science, this issue p. 81, p. 77 A camel vaccine against MERS coronavirus may reduce the risk of human infection and protect against camelpox too. 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.

Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4 Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.

Human picobirnaviruses identified by molecular screening of diarrhea samples.

ABSTRACT The global threat of (re)emerging infectious viruses requires a more effective approach regarding virus surveillance and diagnostic assays, as current diagnostics are often virus species specific and not able to detect highly divergent or unknown viruses. A systematic exploration of viruses that infect humans is the key to effectively counter the potential public health threat caused by new and emerging infectious diseases. The human gut is a known reservoir of a wide variety of microorganisms, including viruses. In this study, Dutch clinical diarrhea samples for which no etiological agent could be identified by available cell culture, serological, or nucleic acid-based tests were gathered. Large-scale molecular RNA virus screening based on host nucleic acid depletion, sequence-independent amplification, and sequencing of partially purified viral RNA from a limited number of clinical diarrhea samples revealed four eukaryotic virus species. Among the detected viruses were a rhinovirus and a new picobirnavirus variant. In total, ∼20% of clinical diarrhea samples contained human picobirnavirus sequences. The Dutch picobirnaviruses belonged to different phylogenetic clades and did not group with other picobirnaviruses according to year of isolation or host species. Interestingly, the average age of patients infected with picobirnavirus was significantly higher than that of uninfected patients. Our data show that sequence-independent amplification of partially purified viral RNA is an efficient procedure for identification of known and highly divergent new RNA viruses in clinical diarrhea samples.

Discontinuous and non‐discontinuous subgenomic RNA transcription in a nidovirus

Arteri‐, corona‐, toro‐ and roniviruses are evolutionarily related positive‐strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona‐ and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity‐assisted copy‐choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative‐strand RNA templates. The sg mRNAs all possess a short 5′ common leader sequence, derived from the 5′ end of the genomic RNA. The joining of the non‐contiguous ‘leader’ and ‘body’ sequences presumably occurs during minus‐strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5′ termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3–5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5′‐terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non‐discontinous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.