•Serological studies on SARS- and MERS-coronavirus (CoV) diagnostics were reviewed.•Different types of serological assays and variable antigens were compared.•Immunogenic epitopes of CoV spike ...proteins were less conserved than nucleocapsid proteins.•Use of spike proteins was found to be superior over nucleocapsid proteins.•Applicability of serological assays for analysis of animal sera was reviewed.
More than a decade after the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002/2003 the occurrence of a novel CoV termed Middle East respiratory syndrome (MERS) CoV challenges researchers and public health authorities. To control spread and finally contain novel viruses, rapid identification and subsequent isolation of infected individuals and their contacts is of utmost importance. Next to methods for nucleic acid detection, validated serological assays are particularly important as the timeframe for antibody detection is less restricted. During the SARS-CoV epidemic a wide variety of serological diagnostic assays were established using multiple methods as well as different viral antigens. Even though the majority of the developed assays showed high sensitivity and specificity, numerous studies reported on cross-reactive antibodies to antigens from wide-spread common cold associated CoVs. In order to improve preparedness and responsiveness during future outbreaks of novel CoVs, information and problems regarding serological diagnosis that occurred during the SARS-CoV should be acknowledged.
In this review we summarize the performance of different serological assays as well as the applicability of the two main applied antigens (spike and nucleocapsid protein) used during the SARS-CoV outbreak. We highlight challenges and potential pitfalls that occur when dealing with a novel emerging coronavirus like MERS-CoV. In addition we describe problems that might occur when animal sera are tested in serological assays for the identification of putative reservoirs. Finally, we give a recommendation for a serological testing scheme and outline necessary improvements that should be implemented for a better preparedness.
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses ...depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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•SARS-CoV-2 uses the SARS-CoV receptor ACE2 for host cell entry•The spike protein of SARS-CoV-2 is primed by TMPRSS2•Antibodies against SARS-CoV spike may offer some protection against SARS-CoV-2
The emerging SARS-coronavirus 2 (SARS-CoV-2) threatens public health. Hoffmann and coworkers show that SARS-CoV-2 infection depends on the host cell factors ACE2 and TMPRSS2 and can be blocked by a clinically proven protease inhibitor. These findings might help to establish options for prevention and treatment.
Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 2019
. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of ...cases with critical courses
. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung
; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 2003
. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission
. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 10
RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic ...assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.
Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 ...(SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.
Summary Background The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus involved in cases and case clusters of severe acute respiratory infection in the Arabian Peninsula, ...Tunisia, Morocco, France, Italy, Germany, and the UK. We provide a full description of a fatal case of MERS-CoV infection and associated phylogenetic analyses. Methods We report data for a patient who was admitted to the Klinikum Schwabing (Munich, Germany) for severe acute respiratory infection. We did diagnostic RT-PCR and indirect immunofluorescence. From time of diagnosis, respiratory, faecal, and urine samples were obtained for virus quantification. We constructed a maximum likelihood tree of the five available complete MERS-CoV genomes. Findings A 73-year-old man from Abu Dhabi, United Arab Emirates, was transferred to Klinikum Schwabing on March 19, 2013, on day 11 of illness. He had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment. The patient died on day 18, due to septic shock. MERS-CoV was detected in two samples of bronchoalveolar fluid. Viral loads were highest in samples from the lower respiratory tract (up to 1·2 × 106 copies per mL). Maximum virus concentration in urine samples was 2691 RNA copies per mL on day 13; the virus was not present in the urine after renal failure on day 14. Stool samples obtained on days 12 and 16 contained the virus, with up to 1031 RNA copies per g (close to the lowest detection limit of the assay). One of two oronasal swabs obtained on day 16 were positive, but yielded little viral RNA (5370 copies per mL). No virus was detected in blood. The full virus genome was combined with four other available full genome sequences in a maximum likelihood phylogeny, correlating branch lengths with dates of isolation. The time of the common ancestor was halfway through 2011. Addition of novel genome data from an unlinked case treated 6 months previously in Essen, Germany, showed a clustering of viruses derived from Qatar and the United Arab Emirates. Interpretation We have provided the first complete viral load profile in a case of MERS-CoV infection. MERS-CoV might have shedding patterns that are different from those of severe acute respiratory syndrome and so might need alternative diagnostic approaches. Funding European Union; German Centre for Infection Research; German Research Council; and German Ministry for Education and Research.
The 2013–2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into ...humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.
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•EBOV adapted to humans during the West African outbreak•Amino acid substitutions in the EBOV glycoprotein increase human cell tropism•The same glycoprotein amino acid substitutions decrease tropism for bat cells
The Ebola virus acquired amino acid substitutions in its glycoprotein that increased its tropism for human cells during the West African outbreak of 2013–2016.
To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the ...past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main camel-exporting countries, Sudan and Somalia, suggesting long-term virus circulation in these animals.
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