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Middle East respiratory syndrome coronavirus (MERS-CoV) causes outbreaks and isolated cases of severe respiratory disease in humans. The virus is transmissible from human to human, but the focus of infection [...]

Middle East respiratory syndrome coronavirus (MERSCoV) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected patients (1,2). Globally, 156 laboratory-confirmed cases of infection with MERS-CoV, [...]

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals (1). However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread (2). Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection (3'4). Viral genome analysis revealed close relatedness to coronaviruses found in bats (5). 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., Coronaviruses infect a wide range of mammals and birds. Their tropism is primarily determined by the ability of the spike (S) entry protein to bind to a cell surface receptor. [...]

Nonparenteral transmission might contribute to human parvovirus 4 (PARV4) infections in sub-Saharan Africa. PARV4 DNA was detected in 8 (0.83%) of 961 nasal samples and 5 (0.53%) of 943 fecal [...]

Abstract Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Background. The Middle East respiratory syndrome (MERS) coronavirus causes isolated cases and outbreaks of severe respiratory disease. Essential features of the natural history of disease are poorly understood. Methods. We studied 37 adult patients infected with MERS coronavirus for viral load in the lower and upper respiratory tracts (LRT and URT, respectively), blood, stool, and urine. Antibodies and serum neutralizing activities were determined over the course of disease. Results. One hundred ninety-nine LRT samples collected during the 3 weeks following diagnosis yielded virus RNA in 93% of tests. Average (maximum) viral loads were 5 × 106 (6 × 1010) copies/mL. Viral loads ( positive detection frequencies) in 84 URT samples were 1.9 × 104 copies/mL (47.6%). Thirty-three percent of all 108 serum samples tested yielded viral RNA. Only 14.6% of stool and 2.4% of urine samples yielded viral RNA. All seroconversions occurred during the first 2 weeks after diagnosis, which corresponds to the second and third week after symptom onset. Immunoglobulin M detection provided no advantage in sensitivity over immunoglobulin G (IgG) detection. All surviving patients, but only slightly more than half of all fatal cases, produced IgG and neutralizing antibodies. The levels of IgG and neutralizing antibodies were weakly and inversely correlated with LRT viral loads. Presence of antibodies did not lead to the elimination of virus from LRT. Conclusions. The timing and intensity of respiratory viral shedding in patients with MERS closely matches that of those with severe acute respiratory syndrome. Blood viral RNA does not seem to be infectious. Extrapulmonary loci of virus replication seem possible. Neutralizing antibodies do not suffice to clear the infection. [ABSTRACT FROM AUTHOR]

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases. [ABSTRACT FROM AUTHOR]

Background: As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results: The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion: The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory infection with as yet unclear epidemiology. We previously showed that MERS-CoV counteracts parts of the innate immune response in human bronchiolar cells. Here we analyzed accessory proteins 3, 4a, 4b, and 5 for their abilities to inhibit the type I interferon response. Accessory protein 4a was found to block interferon induction at the level of melanoma differentiation-associated protein 5 (MDA5) activation presumably by direct interaction with double-stranded RNA. [ABSTRACT FROM AUTHOR]

Bats may host emerging viruses, including coronaviruses (CoV). We conducted an evaluation of CoV in rhinolophid and vespertilionid bat species common in Europe. Rhinolophids carried severe acute respiratory syndrome (SARS)-related CoV at high frequencies and concentrations (26% of animals are positive; up to 2.4 × 108 copies per gram of feces), as well as two Alphacoronavirus clades, one novel and one related to the HKU2 clade. All three clades present in Miniopterus bats in China (HKU7, HKU8, and 1A related) were also present in European Miniopterus bats. An additional novel Alphacoronavirus clade (bat CoV [BtCoV]/BNM98-30) was detected in Nyctalus leisleri. A CoV grouping criterion was developed by comparing amino acid identities across an 816-bp fragment of the RNA-dependent RNA polymerases (RdRp) of all accepted mammalian CoV species (RdRp-based grouping units [RGU]). Criteria for defining separate RGU in mammalian CoV were a >4.8% amino acid distance for alphacoronaviruses and a >6.3% distance for betacoronaviruses. All the above-mentioned novel clades represented independent RGU. Strict associations between CoV RGU and host bat genera were confirmed for six independent RGU represented simultaneously in China and Europe. A SARS-related virus (BtCoV/BM48-31/Bulgaria/2008) from a Rhinolophus blasii (Rhi bla) bat was fully sequenced. It is predicted that proteins 3b and 6 were highly divergent from those proteins in all known SARS-related CoV. Open reading frame 8 (ORF8) was surprisingly absent. Surface expression of spike and staining with sera of SARS survivors suggested low antigenic overlap with SARS CoV. However, the receptor binding domain of SARS CoV showed higher similarity with that of BtCoV/BM48-31/Bulgaria/2008 than with that of any Chinese bat-borne CoV. Critical spike domains 472 and 487 were identical and similar, respectively. This study underlines the importance of assessments of the zoonotic potential of widely distributed bat-borne CoV. [ABSTRACT FROM AUTHOR]