The Coronavirus pandemic has created havoc all around the globe costing many lives and resources in the past few months. With no available cure or vaccine, the only way to stay safe is to stay away from people and maintain good hygiene. Scientists from all over the world are trying their best to develop a vaccine that can eliminate this virus. In the wake of this pandemic as most of us are quarantined at home, it offers an opportunity for us, citizen scientists to learn more about COVID-19 and contribute to the research. Hence the team at Pine Biotech has been organizing a series of Webinars on the Genomics of Covid-19. 

In our recent workshop, our participants asked quite a few interesting questions that we have compiled below:

Q1

Does replication occur only in respiratory cells or all other cells?

Coronavirus enters through mucosal cells (Nose, mouth, eyes), so yes, it can replicate in all epithelial cells. 

Q2

How does the spike cleave into S1 and S2? and where does it cleave?

The Spike protein S1 attaches the virion to the cell membrane by interacting with the host receptor, initiating the infection. Binding to human ACE2, CLEC4M/DC-SIGNR receptors, and internalization of the virus into the endosomes of the host cell induces conformational changes in the S glycoprotein. Proteolysis by a cathepsin named CTSL may unmask the fusion peptide of S2 – and activate membrane fusion within endosomes. 

The Spike protein S2  mediates fusion of the virion and cellular membranes by acting as a class I viral fusion protein. 

Under the current model, the protein has at least three conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. 

During viral & target cell membrane fusion, the coiled coil regions (which are heptad repeats) assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes. 

Spike protein S2′  acts as a viral fusion peptide which is unmasked following S2 cleavage – which occurs upon virus endocytosis.

Q3

What’s the role of fusion peptide? Is it conserved in COVID19 and SARS?

Viral fusion proteins mediate the fusion of membrane-enveloped viruses – such as the influenza virus – with their host cell membranes, so as to release the viral nucleic acids into the host cell cytosol.

• See question 2 above for more information on this process

Q4

What part of the body does this virus attack?

Lung, mainly. Research is going on. It may attack other parts as well.

As with other coronavirus illnesses — including SARS, MERS, and the common cold — COVID-19 is a respiratory disease, so the lungs are usually affected first.

Early symptoms include fever, cough, and shortness of breath. These appear as soon as 2 days, or as long as 14 days, after exposure to the virus.

The severity of COVID-19 varies from mild or no symptoms to severe or sometimes fatal illness. Data on more than 17,000 reported cases in China found that almost 81 percent of cases were mild. The rest were severe or critical.

The lungs are the main organs affected by COVID-19. But in serious cases, the rest of the body can also be affected. However, this can happen with any severe infection. This damage to the organs isn’t always directly caused by the infection, but can result from the body’s response to infection.

Some people with COVID-19 have reported gastrointestinal symptoms, such as nausea or diarrhea, although these symptoms are much less common than problems with the lungs. While coronaviruses seem to have an easier time entering the body through the lungs, the intestines aren’t out of reach for these viruses. Earlier reports identified the viruses that cause SARS and MERS in intestinal tissue biopsies and stool samples.

Two recent studies — one in the New England Journal of Medicine and a preprint on medRxiv — report that stool samples of some people with COVID-19 tested positive for the virus. However, researchers don’t know yet whether fecal transmission of this virus can occur.

COVID-19 can also affect the heart and blood vessels. This may show up as irregular heart rhythms, not enough blood getting to the tissues, or blood pressure low enough that it requires medications. So far, though, there’s no indication that the virus directly damages the heart. With any infection, the body’s immune system responds by attacking the foreign virus or bacteria. While this immune response can rid the body of the infection, it can also sometimes cause collateral damage in the body.

This can come in the form of an intense inflammatory response, sometimes called a “cytokine storm.” The immune cells produce cytokines to fight infection, but if too many are released, it can cause problems in the body.

Q5

A – can you tell how N3 inhibits the main protease?

B – how do N3 inhibitors work ??

You can read more about N3 here and it’s inhibition here

In this figure, you see the COVID-19 main protease that consists of 2 parts (red and blue). On the right, you can see the inhibitor bound to the protein (orange). These are based on the following PDB structures: 6LU7 and 3TLO.

Q6

How did the corona virus come into existence?

Coronaviruses are named for their crown-like shape, and were first identified in the mid-1960s. The virus typically causes respiratory illnesses like the common cold.

In the beginning, many of those infected worked or shopped at a wholesale seafood market in Wuhan, China, which also sold live and freshly slaughtered animals. This is why experts suspect it crossed to humans from an animal host.A new study  found the virus may have originated in bats and then spread to humans via a snake or pangolin. Seven types of coronaviruses are known to infect humans.

https://www.sciencedirect.com/science/article/pii/S2090123220300540#f0010

Q7

 Is the corona virus a living thing?

Like any other virus. (definition of virus: active only inside host cells, outside the host it’s like a particle, inactive. Virus has either RNA or DNA as genome, never both. Covid-19 is an RNA virus.

Q8

What’s 229E?

Human coronavirus 229E is a species of coronavirus which infects humans and bats. The infecting virus is an enveloped, positive-sense, single-stranded RNA virus which enters its host cell by binding to the APN receptor. Along with Human coronavirus OC43, it is one of the viruses responsible for the common cold.

You can read more about 229E here

Q9

What is the first symptom of coronavirus infection mostly ??

Better to ask physicians. Usually pain in throat, shortness of breath, fever, dry cough

Q10

How much similarity is there b/w the spike protein of CoVID-19 and HIV?

They are two very different viruses. HIV has gp41, gp120 and gB 160. 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033698/  

Q11

Is chloroquine really effective? 

It was shown to be effective. https://www.nature.com/articles/s41421-020-0156-0

But administration requires prescription from your doctor. It can be toxic for some patients.

Q12

I wanted to know the difference between phylogeographic and phylogenetic analysis – and if they are different how to perform phylogeographic analysis

Phylogeography is the study of the historical processes that may be responsible for the contemporary geographic distributions of individuals. This is accomplished by considering the geographic distribution of individuals in light of genetics, particularly population genetics. This term was introduced to describe geographically structured genetic signals within and among species. An explicit focus on a species’ biogeography/biogeographical past sets phylogeography apart from classical population genetics and phylogenetics.

In the case of viruses, we can study the viral origin and how it spreads geographically to try to find specific variation linked to location. A good way to see the geographic distribution of covid 19 and the genomic variation in this visualization: https://nextstrain.org/ncov

Q3

What do you think about the possibility of genotyping of new coronaviruses?

You can read more about genotyping coronaviruses here

Q14

Is there any other receptor other than ACE2?

Not known yet. 

Q15

Is there any link between any chemicals like high amounts of Alcl in the body and coronavirus suspected person??

Not known yet. 

Q16

Can nigella sativa be effective against coronavirus?

Not proven

Q17

If we do molecular docking with the protein of coronavirus and known drugs of HIV will it help ?

Drug repurposing has been an approach that many are trying to apply to COVID-19, see more on this approach here: https://www.nature.com/articles/d41586-020-00412-x

There are several steps to this approach – identifying potential active molecules, trying to dock them to various proteins and finally studies on cell lines. The issue with docking is that we do not always know which protein the molecule might bind to.

Q19

Can you use bioinformatics approaches to determine if there could be other receptors in addition to ACE2? Or would that be kind of a waste of time?

There are several published studies that compare host response to viral infection, for example:

J Virol. 2013 Apr;87(7):3885-902. doi: 10.1128/JVI.02520-12. Epub 2013 Jan 30.

Release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells.

Sims AC1, Tilton SC, Menachery VD, Gralinski LE, Schäfer A, Matzke MM, Webb-Robertson BJ, Chang J, Luna ML, Long CE, Shukla AK, Bankhead AR 3rd, Burkett SE, Zornetzer G, Tseng CT, Metz TO, Pickles R, McWeeney S, Smith RD, Katze MG, Waters KM, Baric RS.

Purpose of the experiment was to compare transcriptomics of 2B4 cells (clonal derivative of Calu-3 cells) infected with either icSARS CoV or the icSARS deltaORF6 mutant. Overall design: Calu-3 cells were infected with either icSARS CoV or the icSARS deltaORF6 mutant at MOI of 5.0. Cells samples were collected at 0, 3, 7, 12, 24, 30, 36, 48, 54, 60 or 72h post infection. Each infected sample was done in triplicate. (Triplicates are defined as 3 different wells, plated at the same time using the same cell stock for all replicates.)There are triplicate time-matched mock for each time point from the same cell stock as the rest of samples. Culture medium (the same as what the virus stock is in) was used for the mock infections. The NIAID Systems Virology Center https://www.ncbi.nlm.nih.gov/bioproject/PRJNA149059

Q20

I’ve read that covid won’t be transmitted through blood transfusion. How is it possible if it replicates in all the cells? Or does it replicate only in respiratory cells?

See Question 1

Q21

Chloroquine suppresses the immune response, will we still use it for covid 19 treatment?

See Question 13

Q22

Is passive immunization effective or not for coronavirus victims?

It could be effective. It’s in trial now. 

Q23

Should remdevisir work without problems?

It’s being used now. 

Q24

Can we use drug repurposing for this spike protein or main protease

We can, see Question 17

Q25

Has anyone investigated variants of ACE2 and how they might affect the ability of the virus to bind to cells?

unknown

Q26

Can we use drug repurposing for this spike protein or main protease?

We can, see Question 17

Q27

Why is ELISA not being used for the detection?

Antibody production requires 14-21 days usually, against pathogens. SARS-CoV-2 is a novel virus. We do not know if we produce enough antibodies against it, and how many days it needs or if the antibody level is enough to detect. WE need to detect this virus quickly to confirm whether the patient is positive or not, so that we can isolate them. RT-PCR will detect active replicating virus early enough from the throat swab sample. 

Q28

is transferred…eye contact…and Hair…is it possible…

So far what we know is, transmission occurs through air droplets from a patient through the eyes/nose/mouth of another person. Even if you touch a surface that has the virus, and then if you touch your face (nose,mouth,eyes) you can get infected. 

Q29

What tools are best utilized to develop 3-D modeling in NGS or elsewhere?

NGS provides us with sequence information. We can link it to 3D protein information by the nucleotide-codon-amino acid connection, however we need many more tools to study the 3D structures of proteins.

Q30

Hydroxychloroquine+azithromycin? How effective is it?

Hydroxychloroquine is used to treat malaria. It is also used to prevent malaria infection in areas or regions where it is known that other medicines (eg, chloroquine) may not work.

Hydroxychloroquine belongs to a group of medicines known as antimalarials. It works by preventing or treating malaria, a red blood cell infection transmitted by the bite of a mosquito. However, this medicine is not used to treat severe or complicated malaria.

Hydroxychloroquine is used to treat discoid lupus erythematosus (DLE) or systemic lupus erythematosus (SLE or lupus). It is also used to treat acute and chronic rheumatoid arthritis.

How effective is it? 

Personally, I don’t think this medication is appropriate for COVID-19 treatment. 

Apparently, neither do lupus patients either.

Re. Azithromycin – This drug is an antibiotic that fights bacteria, NOT viruses. Azithromycin is used to treat many different types of infections caused by bacteria, such as respiratory infections, skin infections, ear infections, eye infections, and sexually transmitted diseases.

Like other macrolide antibiotics, azithromycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome.

Q31

What is the function of Nsp?

There are 16 nonstructural peptides, which are called nsps for short. I’ve created a table that lists out the functions of each:

 

nsp1	Promotes cellular mRNA degradation and blocks host cell translation, results in blocking innate immune response
nsp2	No known function, binds to prohibitin proteins
nsp3	Large, multi-domain transmembrane protein, activities include: Ubl1 and Ac domains, interact with N protein ADRP activity, promotes cytokine expression PLPro/Deubiquitinase domain, cleaves viral polyprotein and blocks host innate immune response Ubl2, NAB, G2M, SUD, Y domains, unknown functions
nsp4	Potential transmembrane scaffold protein, important for proper structure of DMVs
nsp5	Mpro, cleaves viral polyprotein
nsp6	Potential transmembrane scaffold protein
nsp7	Forms hexadecameric complex with nsp8, may act as processivity clamp for RNA polymerase
nsp8	Forms hexadecameric complex with nsp7, may act as processivity clamp for RNA polymerase; may act as primase
nsp9	RNA binding protein
nsp10	Cofactor for nsp16 and nsp14, forms heterodimer with both and stimulates ExoN and 2-O-MT activity
nsp12	RdRp
nsp13	RNA helicase, 5′ triphosphatase
nsp14	N7 MTase) and 3′-5′ exoribonuclease, ExoN; N7 MTase adds 5′ cap to viral RNAs, ExoN activity is important for proofreading of viral genome
nsp15	Viral endoribonuclease, NendoU
nsp16	2′-O-MT; shields viral RNA from MDA5 recognition

 

Q32

I’ve read that covid won’t be transmitted through blood transfusion. How is it possible if it replicates in all the cells? Or does it replicate only in respiratory cells?

• When a virus is present in blood it’s called viremia. You CANNOT donate blood that time. 

Q33

What is the quick detection method of Covid-19 in the human body?

RT-PCR – You can read more about the process here

Q34

Is it possible to block the mechanism during the replication process ?

For this you need a specific drug.  One of these uses this approach, and is called Remdesivir. You can read more about this nucleotide analog here

Q35

mRNA based vaccines are under studied. Would you please  tell us about the bioinformatics of mRNA based vaccines?

The idea behind mRNA vaccines is to produce viral proteins using mRNA and not protein directly. The vaccine is now being tested in a clinical trial: https://www.nih.gov/news-events/news-releases/nih-clinical-trial-investigational-vaccine-covid-19-begins

The vaccine is called mRNA-1273 and was developed by NIAID scientists and their collaborators at the biotechnology company Moderna, Inc., based in Cambridge, Massachusetts. The Coalition for Epidemic Preparedness Innovations (CEPI) supported the manufacturing of the vaccine candidate for the Phase 1 clinical trial.

The investigational vaccine directs the body’s cells to express a virus protein that it is hoped will elicit a robust immune response. The mRNA-1273 vaccine has shown promise in animal models, and this is the first trial to examine it in humans.

The bioinformatics approaches involved require in-depth understanding of the novel Coronavirus genomics and how it is different from MERS and SARS.

Q36

How do you see COVID-29 mutating, and have you seen in any mutations in its sequence between January and the present?

It’s very likely. We are waiting for that data. In China there were two strains L and S. 

Read about these here

Q37

Is it possible to obtain vaccines from memory cells of the recovered patients?

The memory cells will tell us what type of immune response is required to fight against covid. But in vaccines,  we need an immunogen that will give us that specific immune response. 

Here is more information on how vaccines work

Q38

Will we have projects on covid-19 in an April workshop which will lead to publications?

We will offer a full hands on program on infectious disease providing easy access to data, tools and interaction with a community of like minded students faculty and researchers.

Read more about it here: https://edu.tbioinfo.com/bioinformatics-for-infectious-diseases-2020

Q39

Are there machine learning strategies for covid-19’s solution?

Machine learning requires a lot of reliable data which at the moment is not largely available, however data from SARS and MERS from host cells can already be useful for predictive modeling, classification and feature selection. Machine Learning can be applied to non genomic data as well.

Q40

What is the E protein?

The envelope protein plays a central role in both virus morphogenesis and assembly. It acts as a viroporin and self-assembles in host membranes forming pentameric protein-lipid pores that allow for ion transport. It also plays a role in the induction of apoptosis. 

The envelope protein also activates the host NLRP3 inflammasome, which leads to IL-1beta overproduction. IL-1 is intensely produced by tissue macrophages, monocytes, fibroblasts, and dendritic cells, but is also expressed by B lymphocytes, NK cells, microglia, and epithelial cells. They form an important part of the inflammatory response of the body against infection. 

They also affect the activity of the hypothalamus, the thermoregulatory center, which leads to a rise in body temperature (fever) . That is why IL-1 is called an endogenous pyrogen. Besides fever, IL-1 also causes hyperalgesia (which is increased pain sensitivity), vasodilation and hypotension.

Q41

Can we remove the spike protein..by using heat on the virus?

• Heat/UV/detergent,soap/alcohol any of these inactivates the lipid envelope of coronavirus.