Background information on SARS-CoV-2 and its detection

Here, we would like to give you an overview of information pages on the current situation regarding SARS-CoV-2 and briefly describe the basics regarding the detection of the pathogen. This information is not only interesting for students specializing in Biochemistry.

(last updated: 12.07.2020, R. Lösel)

Daily updated case numbers in Germany can be viewed on the web pages of the Robert Koch Institute (RKI), while the case numbers worldwide can be obtained by visiting the server of the WHO. The figures from Johns Hopkins University, which evaluates other data sources, may possibly be more up to date. In the meantime, a first version of the so-called Heinsberg study from the Streeck working group has been published, which examines the number of infected persons in a severely affected community after a carnival event. However, the results are not readily transferable to other situations.

A profile of the disease COVID-19 and the pathogen SARS-CoV-2 can be found here (RKI website). The WHO has created a special page (Myth Buster) to clear up frequently spread false reports about COVID-19. Information about protection against infections can also be found on the website of the German Federal Centre for Health Education (BZgA). In the meantime, initial results suggest that it is possible to predict the course of a severe disease using laboratory diagnostics.

Daily updated data on the development of the reproduction number R on the basis of nowcasting as well as the explanation of the models can also be found on the RKI website.

In most countries, the pathogen is detected using a PCR method recommended by the WHO, which is based on an older method for detecting the pathogen that causes SARS (which is also a coronavirus).

These are PCR methods that exploit the 5' nuclease activity of Taq polymerase to degrade a quantized oligonucleotide probe (hybridized to the target sequence). Due to the structure of the probes (fluorescent dye at one end, quencher at the other end) the fluorescence of the dye is quenched by the quencher as long as both are kept in close proximity by the oligonucleotide. As soon as the oligonucleotide is degraded by the 5'-nuclease activity of the polymerase during the new synthesis of a DNA strand and the fluorescent dye and quencher diffuse away from each other, this fluorescence quenching no longer takes place. The intensity of the fluorescence is a direct measure of the amount of newly formed DNA amplificate. A similar experiment is the model experiment Real Time PCR (only accessible via the university network/VPN) in the laboratory course in Diagnostics and Forensics.

The evaluation of the fluorescence curves obtained is described here, for example.

Coronaviruses are medium-sized (+)-stranded RNA viruses that normally cause colds. Further information about their structure and reproduction can be found here.  General information about viruses and their molecular properties can be found in this e-book (only via university network/VPN). A presentation of the mutations of the virus found so far as well as models of global distribution calculated from them is available here.

 

Antibody tests

The detection of antibodies against SARS-CoV2 does not replace direct detection by PCR. Antibodies are only formed following a delay of several days, as they are the reaction of the immune system to the infection. In many causes, the occurrence of antibodies against a pathogen therefore indicates that the infection has been overcome (seroconversion). In the case of SARS-CoV2, however, it is currently unclear how long the immunity to re-infection lasts.

Antibodies are usually quantified by indirect ELISA. Other formats such as rapid tests give only qualitative (yes/no) results. In the case of SARS-CoV2, the problem is that the pathogens of common seasonal colds include many other, more or less related corona viruses. The challenge is therefore to select an antigen that only recognizes antibodies against SARS-CoV2 and not cross-reacting antibodies against other corona viruses. The description of a test system can be found here. In the case of some commercial tests already available, the requirement for specificity does not seem to be fulfilled.

 

Replication of SARS-CoV2

SARS-CoV2 binds with its spike glycoprotein to the angiotensin converting enzyme 2 (ACE2), which is present on the cell surface. After binding, the spike protein is cleaved by the body’s own proteases, the virus is absorbed by the cell and is present in endosomes. From the endosomes/lysosomes, the genome of the virus, a single RNA strand in (+)-orientation, is released into the cytoplasm. From this RNA strand, a new complementary RNA strand is synthesized directly, without detour via DNA. This requires a viral RNA-dependent RNA polymerase (RdRp), which does not occur in non-virus-infected cells. Once sufficient viral proteins for the formation of the capsid have been produced, new virions are formed and leave the host cell.

Experimental active ingredients

Dexamethasone

Dexamethasone is a synthetic, fluorinated glucocorticoid that is about 30 times more effective than natural hydrocortisone. It has been used clinically for more than 50 years. Like all glucocorticoids, dexamethasone suppresses inflammatory reactions, including the cytokine storm that occurs in severe cases of COVID-19 infection. Although dexamethasone does not have a causal effect, i.e. it does not inhibit the replication of the virus, it reduces mortality by about one third and therefore works better than Remdesivir according to some studies.

Chloroquin
Hydroxychloroquin

Quinoline derivatives

Chloroquine (trade name – Resochin) is a relatively old active ingredient produced by the Bayer company and has been used for about 80 years for the therapy and prophylaxis of malaria. Other applications are autoimmune diseases such as rheumatoid arthritis and lupus erythematosus.

In cell culture experiments, both chloroquine and its metabolite hydroxychloroquine (trade name Quensyl) showed effects against both the old SARS coronavirus and SARS-CoV2, the pathogen causing COVID-19. One of the mechanisms of action is probably the change in the pH value in the lysosomes; in addition, endocytosis and thus the absorption of the virus seems to be inhibited. Initial treatment trials on patients showed contradictory results. In view of the numerous, sometimes severe side effects of these active ingredients at the required high doses, the regulatory authorities in Europe, but also in the USA, are critical of their widespread use until more effectively substantiated clinical data are available. The French Food and Drug Administration has issued an explicit warning against certain combinations. The current President of the USA had previously promoted the substance very strongly, describing it as “possibly the greatest breakthrough in medicine”, and in this context, the combination with azithromycin was also publicized. However, this macrolide antibiotic, which blocks protein synthesis on bacterial ribosomes, is ineffective against viruses.

Remdesivir

This compound, which was synthesized by Gilead Sciences and used in experiments during the Ebola epidemic, is a prodrug of the actual active form GS-441524, a C nucleoside that is an analogue of adenosine. In vivo, the nucleoside is phosphorylated into a triphosphate. The mechanism of action is the inhibition of RNA-dependent RNA polymerase (RdRp), which inhibits the replication of the virus. In vitro, the active substance showed broad activity against a number of (+)-strand RNA viruses, including orthomyxo (influenza) and paramyxo (parainfluenza) viruses and some others. In Ebola-RdRp, the incorporation of Remdesivir triphosphate into the RNA primary transcript causes a delayed chain termination, mainly at position i+5.

Although Remdesivir was only moderately effective against Ebola, it is currently considered the most promising candidate and has been used in several clinical phase 3 trials worldwide for the treatment of COVID-19 since March 2020. The manufacturer has promised to be able to produce sufficient quantities to treat more than one million patients by the end of 2020.

Since 3 July 2020, Remdesivir has been approved in the EU for the treatment of some forms of COVID-19.

Remdesivir
GS-441524

Protease inhibitors

Most proteins of the SARS-CoV2 virus are not translated separately, but are formed from a large precursor protein that is cleaved into the various functional viral proteins by a viral protease. This cleaving process is essential. Inhibition of viral proteases has been shown to be effective in the therapy of various viral diseases, especially in the treatment of HIV. A combination of two protease inhibitors (Lopinavir and Ritonavir, trade name Kaletra) is often used for that purpose. In vitro, this combination is also effective against SARS-CoV2, but in an initial clinical study, no significant improvement was observed upon treatment with this protease inhibitor combination.

Lopinavir
Ritonavir

Other active substances

Favipiravir is approved in Japan for the treatment of influenza; in China the substance is considered effective in cases of COVID-19.

The cytokine interferon β-1a acts on the immune system and is also being used experimentally.

The protease inhibitor camostat, which is approved in Japan for the treatment of pancreatitis (inflammation of the pancreas), also blocks the body’s own protease TMPRSS2, which is needed for the virus to enter the cell.

Favipiravir

A summary of other active ingredients and combinations of those currently being used experimentally against SARS-CoV2 can be found here (continuously updated)