scholarly journals Infectious Middle East Respiratory Syndrome Coronavirus Excretion and Serotype Variability Based on Live Virus Isolates from Patients in Saudi Arabia

2015 ◽  
Vol 53 (9) ◽  
pp. 2951-2955 ◽  
Author(s):  
Doreen Muth ◽  
Victor M. Corman ◽  
Benjamin Meyer ◽  
Abdullah Assiri ◽  
Malak Al-Masri ◽  
...  
Keyword(s):  
Middle East ◽  
Infectious Virus ◽  
Virus Isolation ◽  
Vero Cells ◽  
Prototype Strain ◽  
Middle East Respiratory Syndrome ◽  
Serum Samples ◽  
Live Virus ◽  
Virus Isolates ◽  
Rna Concentration

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.

scholarly journals Origins and pathogenesis of Middle East respiratory syndrome-associated coronavirus: recent advances

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1628 ◽  
Author(s):  
Stephen A. Goldstein ◽  
Susan R. Weiss
Keyword(s):  
Immune Response ◽  
Middle East ◽  
Animal Models ◽  
Case Fatality ◽  
Innate Immune ◽  
Middle East Respiratory Syndrome ◽  
Live Virus ◽  
Novel Proteins ◽  
Significant Research ◽  
African Bats

Middle East respiratory syndrome-associated coronavirus (MERS-CoV) has been a significant research focus since its discovery in 2012. Since 2012, 2,040 cases and 712 deaths have been recorded (as of August 11, 2017), representing a strikingly high case fatality rate of 36%. Over the last several years, MERS-CoV research has progressed in several parallel and complementary directions. This review will focus on three particular areas: the origins and evolution of MERS-CoV, the challenges and achievements in the development of MERS-CoV animal models, and our understanding of how novel proteins unique to MERS-CoV counter the host immune response. The origins of MERS-CoV, likely in African bats, are increasingly clear, although important questions remain about the establishment of dromedary camels as a reservoir seeding human outbreaks. Likewise, there have been important advances in the development of animal models, and both non-human primate and mouse models that seem to recapitulate human disease are now available. How MERS-CoV evades and inhibits the host innate immune response remains less clear. Although several studies have identified MERS-CoV proteins as innate immune antagonists, little of this work has been conducted using live virus under conditions of actual infection, but rather with ectopically expressed proteins. Accordingly, considerable space remains for major contributions to understanding unique ways in which MERS-CoV interacts with and modulates the host response. Collectively, these areas have seen significant advances over the last several years but continue to offer exciting opportunities for discovery.

scholarly journals A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

2021 ◽  
Author(s):  
Syed Hani Abidi ◽  
Kehkeshan Imtiaz ◽  
Akbar Kanji ◽  
Shama Qaiser ◽  
Erum Khan ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Neutralization Assay ◽  
Dilution Method ◽  
Rt Pcr ◽  
Serum Samples ◽  
Live Virus ◽  
Viral Neutralization ◽  
Microneutralization Assay ◽  
Infected Cells

Abstract Background Individuals recovering from COVID-19 are shown to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow the determination of virus neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike receptor binding domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.

scholarly journals A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus

2021 ◽  
Vol 12 ◽  
Author(s):  
Kwan Woo Kim ◽  
Sungmi Choi ◽  
Su-Kyoung Shin ◽  
Imchang Lee ◽  
Keun Bon Ku ◽  
...  
Keyword(s):  
Public Health ◽  
Middle East ◽  
Genome Sequencing ◽  
Complete Genome ◽  
Middle East Respiratory Syndrome ◽  
Universal Primers ◽  
Clinical Samples ◽  
Primary Concern ◽  
Whole Genome ◽  
Serum Samples

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

scholarly journals Lack of Middle East Respiratory Syndrome Coronavirus Transmission in Rabbits

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 381 ◽  
Author(s):  
W. Widagdo ◽  
Nisreen M. A. Okba ◽  
Mathilde Richard ◽  
Dennis de Meulder ◽  
Theo M. Bestebroer ◽  
...  
Keyword(s):  
Middle East ◽  
Animal Species ◽  
Infectious Virus ◽  
Clinical Manifestations ◽  
Middle East Respiratory Syndrome ◽  
Viral Rna ◽  
Nasal Discharge ◽  
Upper Respiratory Tract ◽  
Transmission Potential ◽  
Livestock Animal

Middle East respiratory syndrome coronavirus (MERS-CoV) transmission from dromedaries to humans has resulted in major outbreaks in the Middle East. Although some other livestock animal species have been shown to be susceptible to MERS-CoV, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. In this study, we used rabbits to further characterize the transmission potential of MERS-CoV. In line with the presence of MERS-CoV receptor in the rabbit nasal epithelium, high levels of viral RNA were shed from the nose following virus inoculation. However, unlike MERS-CoV-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. Consistently, no transmission by contact or airborne routes was observed in rabbits. Our data indicate that despite relatively high viral RNA levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission.

scholarly journals Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Dromedary Camels in Africa and Middle East

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 717 ◽  
Author(s):  
Kandeil ◽  
Gomaa ◽  
Nageh ◽  
Shehata ◽  
Kayed ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Middle East ◽  
Longitudinal Studies ◽  
Middle East Respiratory Syndrome ◽  
East African ◽  
Dromedary Camels ◽  
Serum Samples ◽  
African Countries ◽  
Nasal Swabs ◽  
Clade C

: Dromedary camels are the natural reservoirs of the Middle East respiratory syndrome coronavirus (MERS-CoV). Camels are mostly bred in East African countries then exported into Africa and Middle East for consumption. To understand the distribution of MERS-CoV among camels in North Africa and the Middle East, we conducted surveillance in Egypt, Senegal, Tunisia, Uganda, Jordan, Saudi Arabia, and Iraq. We also performed longitudinal studies of three camel herds in Egypt and Jordan to elucidate MERS-CoV infection and transmission. Between 2016 and 2018, a total of 4027 nasal swabs and 3267 serum samples were collected from all countries. Real- time PCR revealed that MERS-CoV RNA was detected in nasal swab samples from Egypt, Senegal, Tunisia, and Saudi Arabia. Microneutralization assay showed that antibodies were detected in all countries. Positive PCR samples were partially sequenced, and a phylogenetic tree was built. The tree suggested that all sequences are of clade C and sequences from camels in Egypt formed a separate group from previously published sequences. Longitudinal studies showed high seroprevalence in adult camels. These results indicate the widespread distribution of the virus in camels. A systematic active surveillance and longitudinal studies for MERS-CoV are needed to understand the epidemiology of the disease and dynamics of viral infection.

scholarly journals Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Fernando Almazán ◽  
Marta L. DeDiego ◽  
Isabel Sola ◽  
Sonia Zuñiga ◽  
Jose L. Nieto-Torres ◽  
...  
Keyword(s):  
Middle East ◽  
Mortality Rate ◽  
Bacterial Artificial Chromosome ◽  
Cdna Clone ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Vaccine Candidate ◽  
Artificial Chromosome ◽  
Middle East Respiratory Syndrome ◽  
E Protein

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein intrans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.IMPORTANCESince the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein intrans, whereas it would only survive a single virus infection cyclein vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

scholarly journals Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels in Nigeria, 2015

2015 ◽  
Vol 20 (49) ◽  
Author(s):  
Daniel KW Chu ◽  
Jamiu O Oladipo ◽  
Ranawaka APM Perera ◽  
Sulaiman A Kuranga ◽  
Samuel MS Chan ◽  
...  
Keyword(s):  
Middle East ◽  
Arabian Peninsula ◽  
Phylogenetic Analyses ◽  
Quantitative Polymerase Chain Reaction ◽  
Middle East Respiratory Syndrome ◽  
Dromedary Camels ◽  
Chain Reaction ◽  
Serum Samples ◽  
Nasal Swabs ◽  
Polymerase Chain

Evidence of current and past Middle East respiratory syndrome coronavirus (MERS-CoV) infection in dromedary camels slaughtered at an abattoir in Kano, Nigeria in January 2015, was sought by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and serology. MERS-CoV RNA was detected in 14 (11%) of 132 nasal swabs and antibody in 126 (96%) of 131 serum samples. Phylogenetic analyses demonstrate that the viruses in Nigeria are genetically distinct from those reported in the Arabian peninsula.

scholarly journals Investigation of MERS-CoV seropositivity among Umrah visitors from the Çorum Region of Turkey

2021 ◽  
Vol 38 (4) ◽  
pp. 608-612
Author(s):  
Ayşe Semra GÜRESER ◽  
Derya YAPAR ◽  
Özlem AKDOĞAN ◽  
Ayşegül TAYLAN ÖZKAN ◽  
Nurcan BAYKAM
Keyword(s):  
Saudi Arabia ◽  
Middle East ◽  
Respiratory Tract ◽  
Middle East Respiratory Syndrome ◽  
Control Group ◽  
Upper Respiratory Tract ◽  
Serum Samples ◽  
Training And Research ◽  
Group Members ◽  
Positive Results

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes Middle East Respiratory Syndrome (MERS). Since the vast majority of cases (more than 85%) are reported from Saudi Arabia, there is a pandemic potential for pilgrimage due to Hajj or Umrah. It is reported from Turkey that more than 400 thousand people went to Saudi Arabia for umrah and 61 thousand people for Hajj in 2014. In this study it is aimed to investigate the patients who had just returned from Makkah for Umrah and who also applied to the Infectious Disease Clinics at the Hitit University Erol Olcok Training and Research Hospital for having respiratory tract symptoms. Their serologic situations have been determined by ELISA whether there is any risk in terms of performing the Hajj and Umrah, and contracting MERS-CoV. Between January 1st to the 31st of October 2015, 40 people were included in this study, which were admitted to our hospital with upper respiratory tract complaints and had previously been in Saudi Arabia for Umrah within the last 15 days. As a control group, 40 healthy people without any complaints and travel histories to risky areas were selected. Their serum samples were taken and searched by MERS-CoV IgG ELISA (Euroimmun AG, Lübeck, Germany). The results ≤0.8 were considered as negative, ≥1.1 were as positive, 0.8-1.1 were suspected. All suspected and positive results have been revaluated and confirmed. Only two (5%) individuals from the patients’ group were found as positive for the MERS-CoV IgG antibodies, but individuals from the remaining patients’ group and also all control group members were determined as negative. Travels to Saudi Arabia could be a risk for exposure to MERS-CoV. Although there is no evidence, contamination could be realized by anthropologically due to crowds.

scholarly journals A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0259551
Author(s):  
Syed Hani Abidi ◽  
Kehkashan Imtiaz ◽  
Akbar Kanji ◽  
Shama Qaiser ◽  
Erum Khan ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Neutralization Assay ◽  
Dilution Method ◽  
Rt Pcr ◽  
Serum Samples ◽  
Live Virus ◽  
Viral Neutralization ◽  
Microneutralization Assay ◽  
Infected Cells

Background Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro‐neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

scholarly journals Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

2013 ◽  
Vol 87 (23) ◽  
pp. 12552-12561 ◽  
Author(s):  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Shutoku Matsuyama
Keyword(s):  
Middle East ◽  
Cell Surface ◽  
Serine Protease ◽  
Virus Entry ◽  
Cathepsin L ◽  
Vero Cells ◽  
Middle East Respiratory Syndrome ◽  
Simultaneous Treatment ◽  
Virus Growth ◽  
Transmembrane Serine Protease

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

Sign in / Sign up

Export Citation Format

Share Document