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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to ...clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome
. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary Background Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of ...infections in people who have had contact with camels is unknown and most index patients cannot recall any such contact. We aimed to do a nationwide serosurvey in Saudi Arabia to establish the prevalence of MERS-CoV antibodies, both in the general population and in populations of individuals who have maximum exposure to camels. Methods In the cross-sectional serosurvey, we tested human serum samples obtained from healthy individuals older than 15 years who attended primary health-care centres or participated in a national burden-of-disease study in all 13 provinces of Saudi Arabia. Additionally, we tested serum samples from shepherds and abattoir workers with occupational exposure to camels. Samples were screened by recombinant ELISA and MERS-CoV seropositivity was confirmed by recombinant immunofluorescence and plaque reduction neutralisation tests. We used two-tailed Mann Whitney U exact tests, χ2 , and Fisher's exact tests to analyse the data. Findings Between Dec 1, 2012, and Dec 1, 2013, we obtained individual serum samples from 10 009 individuals. Anti-MERS-CoV antibodies were confirmed in 15 (0·15%; 95% CI 0·09–0·24) of 10 009 people in six of the 13 provinces. The mean age of seropositive individuals was significantly younger than that of patients with reported, laboratory-confirmed, primary Middle Eastern respiratory syndrome (43·5 years SD 17·3 vs 53·8 years 17·5; p=0·008). Men had a higher antibody prevalence than did women (11 0·25% of 4341 vs two 0·05% of 4378; p=0·028) and antibody prevalence was significantly higher in central versus coastal provinces (14 0·26% of 5479 vs one 0·02% of 4529; p=0·003). Compared with the general population, seroprevalence of MERS-CoV antibodies was significantly increased by 15 times in shepherds (two 2·3% of 87, p=0·0004) and by 23 times in slaughterhouse workers (five 3·6% of 140; p<0·0001). Interpretation Seroprevalence of MERS-CoV antibodies was significantly higher in camel-exposed individuals than in the general population. By simple multiplication, a projected 44 951 (95% CI 26 971–71 922) individuals older than 15 years might be seropositive for MERS-CoV in Saudi Arabia. These individuals might be the source of infection for patients with confirmed MERS who had no previous exposure to camels. Funding European Union, German Centre for Infection Research, Federal Ministry of Education and Research, German Research Council, and Ministry of Health of Saudi Arabia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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