Publications by authors named "Stalin Raj"

56 Publications

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment.

J Biol Chem 2020 Nov 5. Epub 2020 Nov 5.

Molecular Biophysics Unit (MBU), Indian Institute of Science, Bangalore, India.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over four weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
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http://dx.doi.org/10.1074/jbc.RA120.016284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832000PMC
November 2020

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development.

EBioMedicine 2020 May 2;55:102743. Epub 2020 Apr 2.

Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, W1148 Biomedical Science Tower, 200 Lothrop St., Pennsylvania, PA 15213, USA. Electronic address:

Background: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection.

Methods: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines.

Findings: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization.

Interpretation: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.
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http://dx.doi.org/10.1016/j.ebiom.2020.102743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128973PMC
May 2020

Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections.

Emerg Infect Dis 2019 10 17;25(10):1868-1877. Epub 2019 Oct 17.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.
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http://dx.doi.org/10.3201/eid2510.190051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759241PMC
October 2019

Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein.

Emerg Microbes Infect 2019 ;8(1):516-530

a Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine , Utrecht University , Utrecht , Netherlands.

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.
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http://dx.doi.org/10.1080/22221751.2019.1597644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455120PMC
June 2019

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection.

Sci Adv 2018 08 8;4(8):eaas9667. Epub 2018 Aug 8.

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

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.
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http://dx.doi.org/10.1126/sciadv.aas9667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082650PMC
August 2018

Middle East respiratory syndrome coronavirus specific antibodies in naturally exposed Israeli llamas, alpacas and camels.

One Health 2018 Jun 4;5:65-68. Epub 2018 May 4.

Kimron Veterinary Institute, Bet Dagan, Israel.

Thus far, no human MERS-CoV infections have been reported from Israel. Evidence for the circulation of MERS-CoV in dromedaries has been reported from almost all the countries of the Middle East, except Israel. Therefore, we aimed to analyze MERS-CoV infection in Israeli camelids, sampled between 2012 and 2017. A total of 411 camels, 102 alpacas and 19 llamas' sera were tested for the presence of antibodies to MERS-CoV. Our findings indicate a lower MERS-CoV seropositivity among Israeli dromedaries than in the surrounding countries, and for the first time naturally infected llamas were identified. In addition, nasal swabs of 661 camels, alpacas and lamas, obtained from January 2015 to December 2017, were tested for the presence of MERS-CoV RNA. All nasal swabs were negative, indicating no evidence for MERS-CoV active circulation in these camelids during that time period.
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http://dx.doi.org/10.1016/j.onehlt.2018.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000904PMC
June 2018

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.

Proc Natl Acad Sci U S A 2017 10 18;114(40):E8508-E8517. Epub 2017 Sep 18.

Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands;

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1 through S1 Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1 We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1 When multivalently displayed on nanoparticles, S1 or S1 bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5--glycolylation and (7,)9--acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.
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http://dx.doi.org/10.1073/pnas.1712592114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635925PMC
October 2017

Phenotypic Differences between Asian and African Lineage Zika Viruses in Human Neural Progenitor Cells.

mSphere 2017 Jul-Aug;2(4). Epub 2017 Jul 26.

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

Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells-which are important target cells in the development of congenital microcephaly-less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.
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http://dx.doi.org/10.1128/mSphere.00292-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555676PMC
July 2017

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

J Virol 2017 11 13;91(21). Epub 2017 Oct 13.

Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals and Health, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication , and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth ; (iii) seven of the nonessential proteins affect viral growth , and two affect virulence ; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.
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http://dx.doi.org/10.1128/JVI.01209-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640863PMC
November 2017

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

MERS-coronavirus: From discovery to intervention.

One Health 2017 Jun 23;3:11-16. Epub 2016 Dec 23.

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

Middle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans and animals affected by this virus and propose specific intervention strategies that would be appropriate to control MERS-CoV. One-third of MERS-CoV patients develop severe lower respiratory tract infection and succumb to a fatal outcome; these patients would require effective therapeutic antiviral therapy. Because of the lack of such intervention strategies, supportive care is the best that can be offered at the moment. Limiting viral spread from symptomatic human cases to health care workers and family members, on the other hand, could be achieved through prophylactic administration of MERS-CoV neutralizing antibodies and vaccines. To ultimately prevent spread of the virus into the human population, however, vaccination of dromedary camels - currently the only confirmed animal host for MERS-CoV - may be the best option to achieve a sustained drop in human MERS cases in time. In the end, a One Health approach combining all these different efforts is needed to tackle this zoonotic outbreak.
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http://dx.doi.org/10.1016/j.onehlt.2016.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454172PMC
June 2017

Genetic diversity of hepatitis C virus in Ethiopia.

PLoS One 2017 1;12(6):e0179064. Epub 2017 Jun 1.

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

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453619PMC
September 2017

Middle East respiratory syndrome coronavirus vaccines: current status and novel approaches.

Curr Opin Virol 2017 04 13;23:49-58. Epub 2017 Apr 13.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands. Electronic address:

Middle East respiratory syndrome coronavirus (MERS-CoV) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. The re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. Ongoing efforts to develop MERS-CoV vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. Extending on our current knowledge of MERS, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish MERS-CoV transmission to humans. In addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next CoV outbreak.
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http://dx.doi.org/10.1016/j.coviro.2017.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102752PMC
April 2017

Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus.

Emerg Infect Dis 2017 02 15;23(2):232-240. Epub 2017 Feb 15.

Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs. To determine whether other animals are potential reservoirs, we inoculated MERS-CoV into llamas, pigs, sheep, and horses and collected nasal and rectal swab samples at various times. The presence of MERS-CoV in the nose of pigs and llamas was confirmed by PCR, titration of infectious virus, immunohistochemistry, and in situ hybridization; seroconversion was detected in animals of both species. Conversely, in sheep and horses, virus-specific antibodies did not develop and no evidence of viral replication in the upper respiratory tract was found. These results prove the susceptibility of llamas and pigs to MERS-CoV infection. Thus, the possibility of MERS-CoV circulation in animals other than dromedaries, such as llamas and pigs, is not negligible.
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http://dx.doi.org/10.3201/eid2302.161239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324816PMC
February 2017

A novel hepatitis B virus subgenotype D10 circulating in Ethiopia.

J Viral Hepat 2017 02 3;24(2):163-173. Epub 2016 Nov 3.

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

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa.
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http://dx.doi.org/10.1111/jvh.12631DOI Listing
February 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

Deletion Variants of Middle East Respiratory Syndrome Coronavirus from Humans, Jordan, 2015.

Emerg Infect Dis 2016 Apr;22(4):716-9

We characterized Middle East respiratory syndrome coronaviruses from a hospital outbreak in Jordan in 2015. The viruses from Jordan were highly similar to isolates from Riyadh, Saudi Arabia, except for deletions in open reading frames 4a and 3. Transmissibility and pathogenicity of this strain remains to be determined.
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http://dx.doi.org/10.3201/eid2204.152065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806954PMC
April 2016

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels.

J Virol 2016 May 14;90(9):4838-4842. Epub 2016 Apr 14.

Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands

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.
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http://dx.doi.org/10.1128/JVI.02994-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836314PMC
May 2016

Cross host transmission in the emergence of MERS coronavirus.

Curr Opin Virol 2016 Feb 29;16:55-62. Epub 2016 Jan 29.

Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands. Electronic address:

Coronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact that closely related viruses have been isolated from this but not any other animal species. Given the widespread geographical distribution of dromedaries found seropositive for MERS-CoV, continued transmission may likely occur in the future. Therefore, a further understanding of the cross host transmission of MERS-CoV is needed to limit the risks this virus poses to man.
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http://dx.doi.org/10.1016/j.coviro.2016.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102731PMC
February 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

Occupational Exposure to Dromedaries and Risk for MERS-CoV Infection, Qatar, 2013-2014.

Emerg Infect Dis 2015 Aug;21(8):1422-5

We determined the presence of neutralizing antibodies to Middle East respiratory syndrome coronavirus in persons in Qatar with and without dromedary contact. Antibodies were only detected in those with contact, suggesting dromedary exposure as a risk factor for infection. Findings also showed evidence for substantial underestimation of the infection in populations at risk in Qatar.
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http://dx.doi.org/10.3201/eid2108.150481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517733PMC
August 2015

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

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

Virological and serological analysis of a recent Middle East respiratory syndrome coronavirus infection case on a triple combination antiviral regimen.

Int J Antimicrob Agents 2014 Dec 18;44(6):528-32. Epub 2014 Sep 18.

Department of Microbiology, University of Athens, Medical School, 75 Mikras Asias Street, 115 27 Athens, Greece. Electronic address:

Serological, molecular and phylogenetic analyses of a recently imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) in Greece are reported. Although MERS-CoV remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected 2 days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from Day 13 of illness. Phylogenetic analysis of the virus showed close similarity with other human MERS-CoVs from the recent Jeddah outbreak in Saudi Arabia. Immunoglobulin G (IgG) titres peaked 3 weeks after the onset of illness, whilst IgM levels remained constantly elevated during the follow-up period (second to fifth week of illness). Serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient.
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http://dx.doi.org/10.1016/j.ijantimicag.2014.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127532PMC
December 2014

Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice.

Vaccine 2014 Oct 3;32(45):5975-82. Epub 2014 Sep 3.

Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA. Electronic address:

A new type of coronavirus has been identified as the causative agent underlying Middle East Respiratory Syndrome (MERS). The MERS coronavirus (MERS-CoV) has spread in the Middle East, but cases originating in the Middle East have also occurred in the European Union and the USA. Eight hundred and thirty-seven cases of MERS-CoV infection have been confirmed to date, including 291 deaths. MERS-CoV has infected dromedary camel populations in the Middle East at high rates, representing an immediate source of human infection. The MERS-CoV spike (S) protein, a characteristic structural component of the viral envelope, is considered as a key target of vaccines against coronavirus infection. In an initial attempt to develop a MERS-CoV vaccine to ultimately target dromedary camels, we constructed two recombinant adenoviral vectors encoding the full-length MERS-CoV S protein (Ad5.MERS-S) and the S1 extracellular domain of S protein (Ad5.MERS-S1). BALB/c mice were immunized with both candidate vaccines intramuscularly and boosted three weeks later intranasally. All the vaccinated animals had antibody responses against spike protein, which neutralized MERS-CoV in vitro. These results show that an adenoviral-based vaccine can induce MERS-CoV-specific immune responses in mice and hold promise for the development of a preventive vaccine that targets the animal reservoir, which might be an effective measure to eliminate transmission of MERS-CoV to humans.
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http://dx.doi.org/10.1016/j.vaccine.2014.08.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115510PMC
October 2014