Skip to main content

Latest publications

Sep 1st, 2016
Chapter Two – Coronavirus Spike Protein and Tropism Changes. Advances in Virus Research
Hulswit RJG, de Haan CAM, Bosch BJ

Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-CoV. Due to their inherent genetic variability, it is inevitable that new cross-species transmission events of these enveloped, positive-stranded RNA viruses will occur. Research into these medical and veterinary important pathogens—sparked by the SARS and MERS outbreaks—revealed important principles of inter- and intraspecies tropism changes. The primary determinant of CoV tropism is the viral spike (S) entry protein. Trimers of the S glycoproteins on the virion surface accommodate binding to a cell surface receptor and fusion of the viral and cellular membrane. Recently, high-resolution structures of two CoV S proteins have been elucidated by single-particle cryo-electron microscopy. Using this new structural insight, we review the changes in the S protein that relate to changes in virus tropism. Different concepts underlie these tropism changes at the cellular, tissue, and host species level, including the promiscuity or adaptability of S proteins to orthologous receptors, alterations in the proteolytic cleavage activation as well as changes in the S protein metastability. A thorough understanding of the key role of the S protein in CoV entry is critical to further our understanding of virus cross-species transmission and pathogenesis and for development of intervention strategies

96:29-57. doi: 10.1016/bs.aivir.2016.08.004
Advances in Virus Research
Jul 15th, 2016
Virulence factors in porcine coronaviruses and vaccine design
Zuñiga S, Pascual-Iglesias A, Sanchez CM, Sola I, Enjuanes L.

Porcine enteric coronaviruses (CoVs) cause severe disease in the porcine herds worldwide, leading to important economic losses. Despite the knowledge of these viruses since the 1970s, vaccination strategies have not been implemented, leading to continuous re-emergence of novel virulent strains. Live attenuated vaccines historically have been the most efficient. We consider that the new trend is the development of recombinant vaccines by using reverse genetics systems to engineer attenuated viruses, which could be used as effective and safe modified live vaccine candidates. To this end, host cell signaling pathways influencing porcine CoV virulence should be identified. Similarly, the identity of viral proteins involved in the modulation of host cell pathways influencing CoV pathogenesis should be analyzed. With this information, and using reverse genetics systems, it is possible to design viruses with modifications in the viral proteins acting as virulence factors, which may lead to attenuated viruses and, therefore, vaccine candidates. In addition, novel antiviral drugs may be developed once the host cell pathways and the molecular mechanism affecting porcine CoV replication and virulence are known. This review is focused in the host cell responses to enteric porcine CoV infection and the viral proteins involved in pathogenesis.

226:142-151. doi:10.1016/j.virusres.2016.07.003
Virus Research
Mar 23rd, 2016
Mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response
Becares M, Pascual-Iglesias A, Nogales A, Sola I, Enjuanes L, Zuñiga S

Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus. Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.

90:5399–5414. doi:10.1128/JVI.03259-15
J Virol
Mar 16th, 2016
Mutations in the Schmallenberg virus Gc glycoprotein facilitate cellular protein synthesis shutoff and restore pathogenicity of NSs deletion mutants in mice
Varela M, Pinto RM, Caporale M, Piras IM, Taggart A, Seehusen F, Hahn K, Janowicz A, de Souza WM, Baumgärtner W, Shi X, Palmarini M

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein.

90:5440–5450. doi:10.1128/JVI.00424-16
J Virol
Feb 17th, 2016
Differential expression of the Middle East respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels.
Widagdo W, Raj VS, Schipper D, Kolijn K, van Leenders GJLH, Bosch BJ, Bensaid A, Segalés J, Baumgärtner W, Osterhaus ADME, Koopmans MP, van den Brand JMA, Haagmans BL

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor—dipeptidyl peptidase 4 (DPP4)—is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.

90:4838–4842. doi:10.1128/JVI.02994-15
J Virol
Jan 1st, 2016
An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels
Haagmans BL, van den Brand JMA, Raj VS, Volz A, Wohlsein P, Smits SL, Schipper D, Bestebroer TM, Okba N, Fux R, Bensaid A, Solanes Foz D, Kuiken T, Baumgärtner W, Segalés J, Sutter G, Osterhaus ADME

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

351(6268):77-81. doi:10.1126/science.aad1283
Science
Feb 1st, 2016
Neurotropic virus infections as the cause of immediate and delayed neuropathology
Ludlow, M., Kortekaas, J., Herden, C. et al

A wide range of viruses from different virus families in different geographical areas, may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the central nervous system, frequently leaving the patient or affected animal with a poor or fatal prognosis. Mechanisms that govern neuropathogenesis and immunopathogenesis of viral infections are highlighted, using examples of well-studied virus infections that are associated with these alterations in different populations throughout the world. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of mechanisms that underlie their clinical manifestations may be expected to provide tools for the development of more effective intervention strategies and treatment regimens.

131: 159. doi:10.1007/s00401-015-1511-3
Acta Neuropathol
Oct 29th, 2015
Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.
Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, et al.

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

11(10): e1005215. doi:10.1371/journal.ppat.1005215
PLoS Pathog
Sep 1st, 2015
Infectious Middle East respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in Saudi Arabia
Muth D, Corman VM, Meyer B, Assiri A, Al-Masri M, Farah M, Steinhagen K, Lattwein E, Al-Tawfiq JA, Albarrak A, Müller MA, Drosten C, Memish ZA

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 1,082 people, including 439 fatalities. So far, no empirical virus isolation study has been done to elucidate infectious virus secretion or serotype variability. Here, we used 51 respiratory samples from 32 patients with confirmed MERS-CoV infection for virus isolation in Vero B4 and Caco-2 cells. We found Caco-2 cells to significantly enhance isolation success over routinely used Vero cells. Isolation success correlated with viral RNA concentration and time after diagnosis as well as with the amount of IgA antibodies secreted in respiratory samples used for isolation. Results from plaque reduction neutralization assays using a representative range of serum samples and virus isolates suggested that all circulating human MERS-CoV strains represent one single serotype. The choice of prototype strain is not likely to influence the success of candidate MERS-CoV vaccines. However, vaccine formulations should be evaluated for their potential to induce IgA.

53:2951–2955. doi:10.1128/JCM.01368-15
J Clin Microbiol
Dec 18th, 2015
Camel vaccine offers hope to stop MERS
Kai Kupferschmidt

Since it was discovered in 2012, the camel-borne virus causing Middle East respiratory syndrome (MERS) has sickened more than 1200 people in the Middle East and killed more than 500 of them.

Scientists worry that the slow-burning epidemic could turn into a global pandemic if the virus changes, so they're hard at work on candidate vaccines for people. But to stamp out the virus before it escalates into an emergency, says Christian Drosten, a virologist at the University of Bonn in Germany, "the best strategy is trying to suppress circulation of the virus in camels." This week online in Science a team reports encouraging results for one candidate camel vaccine, though its main effect—reducing the level of virus shed by the animals—may not be enough to stop MERS's circulation. Nor is it clear whether the vaccine gives lasting protection or whether camel owners will accept vaccinating against a disease that causes hardly any symptoms in the animals.

Vol. 350, Issue 6267, pp. 1453, DOI: 10.1126/science.350.6267.1453
Science

Pages

Main menu