Projections and panic aside let’s dissect the history, mechanism, genetics and pathology of this virus. A number of different names are in circulation describing it – 2019-nCoV and SARS-CoV-2 – with the underlying condition it causes termed COVID-19. 2019-nCoV was the official name given to the novel coronavirus by the WHO, however SARS-CoV-2 is the name used in many studies to reflect on the similarities and differences between SARS-CoV (responsible for the 2002 SARS outbreak) and the novel SARS-CoV-2. I will use these names interchangeably but prefer the term 2019-nCoV as it leaves no room for confusion between the novel coronavirus and the SARS-causing strain.
The first member of the coronavirus family was discovered in the 1930s through reports of viral bronchitis . The SARS-CoV (severe acute respiratory syndrome) outbreak in 2002-2003 led to the identification of many new family members of the coronavirus and also proved its ability to jump between species. Prior to the discovery of SARS, coronaviruses were largely of veterinary interest where they were known to cause respiratory and enteric diseases in a wide variety of mammals and birds. SARS-CoV shares 79.5% sequence identity with the novel coronavirus SARS-CoV-2 that causes COVID-19.
It is thought that the new strain of 2019-nCoV, originating from Wuhan, China was transmitted from an animal source to humans. Doctors came to this conclusion early in December 2019 after conducting a survey on patients presenting with 2019-nCoV in Wuhan. Out of the first 41 patients originally confirmed with 2019-nCoV, they found that 27 (66%) had been exposed to Huanan seafood market. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Phylogenetic analysis has shown the 2019-nCoV sequence to be most closely related to bat coronaviruses of the Betacoronaviruses genera. CoVZC45 and CoVZXC21 (marked in orange on the tree below) are strains found in bats and are recombinants of 2019-nCoV with shared break points at the ORF1b (open reading frame) gene.
Recombinants occur by crossing over of DNA or RNA strands during meiosis (cell division) or by reassortment of genomic segments. Alongside mutation in replication of the RNA strand, recombination is a driving force of evolution in viruses.
Although this phylogenetic reconstruction shows that the bat viruses CoVZC45 and CoVZXC21 are recombinants of the 2019-nCoV virus, the author of this study emphasizes that it is not conclusive evidence that 2019-nCoV originated directly in a bat intermediate and that other species cannot be disregarded. We do know however that 2019-nCoV shares greater than 96% sequence similarity with that of a bat virus detected in 2013.
Interestingly, another analysis showed 97% sequence similarity between the 2019-nCoV’s spike glycoprotein binding domain with the same binding region of a coronavirus isolated from Malayan pangolins.
We’ve been bombarded with images of a spherical virus with many formidable protruding spike appendages. These are called spike glycoproteins (S) and are characteristic of coronaviruses. The spike glycoprotein is what facilitates binding to the ACE-2 (angiotensin converting enzyme) receptor on host cells and mediates entry through fusion of the host and viral membranes.
Changes in conformation of the S glycoprotein, a class I fusion protein, enables the different stages of virus entry, cell tropism (which cells and tissues it can enter and infect) and pathogenesis.
Spike proteins assemble into trimers on the virus surface which give the virus its distinctive “corona” or crown-like appearance. This large type I transmembrane protein is 1000-1400 AAs in length.
The spike (S) protein has a further 2 subunits – the surface unit S1 responsible for viral attachment to host cells, and the S2 subunit which drives fusion of the viral and host cell membranes after being cleaved by cellular proteases.
In addition to the S protein which is mediator of host cell entry and determining which host cells it can bind to and enter, the viral envelope has a membrane protein (M) and an envelope protein (E) which are involved in viral assembly and reproduction inside host cells .
Coronaviruses range from 80 to 120nm in size. For size comparison a human hair is around 75μm (microns) which is 75,000nm. Their RNA genome is of length between 26.2 and 31.7kb, the longest among all RNA viruses. Packaged into a helical nucleocapsid, their RNA genome is surrounded by a host-derived lipid bilayer.
Viral entry and pathogenesis
Using their sophisticated S glycoprotein, coronaviruses are able to exploit many cell surface molecules to gain entry into target cells. Viral entry can occur at the host cell surface through binding and fusion of a host cell receptor or after internalization via endocytosis with fusion taking place in the endosomal compartment. Endosomal compartments are lipid bubbles or sacs which temporarily store the newly entered cell contents or visitor until their fate is determined.
By binding to our host cells ACE2 receptors, 2019-nCoV not only gains internal entry but may trigger their physiological functions as a receptor agonist.
The virus is an opportunistic pirate and initiates a hostile takeover of the cell once on board. The viral nsp1 protein initiates degradation of host cell mRNA and blocks host cell translation which results in completely disarming and halting our cell’s innate immune response. Our innate immune response can be thought of as our front-line defense which sounds the alarm when an intruder has arrived as well as recruiting the rest of our immune artillery for back-up.
While the nsp1 protein has successfully neutralised our immune intelligence, a number of other viral subunits set to work directing the mass reproduction and assembly of virus parts by hijacking host cell pathways and machinery.
A full list of subunit functions can be viewed here. Next it sets to work replicating many copies of its viral RNA, the genetic sequence containing the blueprint for the creation of more coronaviruses.
Once RNA replication is complete, the S, E and M structural proteins are translated from the RNA. These structural proteins are inserted into the endoplasmic reticulum (ER) and migrate through this protein production factory where viral genomes will bud off the ER membranes becoming encapsulated with the S, E and M functional proteins. The newly assembled viral spawn are shuttled by golgi vesicles to the host’s cell membrane for ejection outside the cell ready to attack new unsuspecting cellular targets.
The entry of SARS‐CoV (2002) into human host cells is mediated mainly by the cell receptor angiotensin‐converting enzyme 2 (ACE2), which is expressed in human airway epithelia, lung parenchyma, vascular endothelia, kidney cells, and small intestine cells
The primary cell receptor 2019-nCoV targets to enter the cell is angiotensin-converting enzyme 2 (ACE2) which is most abundantly expressed in lung tissue. ACE2 are expressed on type I and II pneumocytes (alveoli cells), ciliated bronchial epithelial cells and small intestine cells.
Tissues of the upper respiratory tract, such as oral and nasal mucosa and nasopharynx, did not show ACE2 expression on the surface of epithelial cells, suggesting that these tissues are not the primary site of entrance for SARS-CoV or HCoV-NL63. Membrane ectopeptidases targeted by human coronaviruses.
A recent study has found that nCOV-2019’s S glycoprotein binds to ACE2 with greater affinity than SARS-CoV. The ACE2 receptor is an important regulator in the renin–angiotensin–aldosterone system (RAAS) pathway. The RAAS is a tightly regulated hormonal feedback system which regulates blood pressure, fluid and electrolyte balance and vascular resistance (blood flow).
Angiotensin II, a product of the RAAS pathway, has potent vasoconstrictive, pro-inflammatory, and pro-fibrotic properties and is inactivated by ACE2. By binding of the 2019-nCOV S protein to ACE2, a physiological effect ensues by down-regulating Angiotensin II’s enzymatic activity.
EDIT: For a more detailed coverage of this pathway check out Medcram’s video:
Experimental models in mice where the ACE2 gene was knocked out have shown it plays a crucial role for protecting against acute respiratory distress syndrome (ARDS), the kind of pathology commonly observed in patients with SARS or avian influenza A. In these mice without ACE2 receptors they found a decrease in lung elasticity, decreased oxygenated blood in the lungs, and the development of pulmonary oedema (increased fluid in the lungs).
2019-nCoV is similarly causing severe acute lung failure through a hardening of the lungs tissues restricting the flow of oxygenated blood into the lungs.
A genetic analysis has already been done to examine if ACE2 expression differs between populations with findings showing that East Asian populations have a higher expression than others.
Gastrointestinal ACE2 expression
Recent bioinformatics analysis on normal human lung and gastrointestinal system transcriptomes (the sum of all mRNA expression) revealed that ACE2 is not only highly expressed in lung cells, but also in the esophagus upper, and in epithelial cells and absorptive enterocytes in the ileum and colon.
This means our digestive system may be vulnerable to SARS-CoV2. As clinicians have been largely focused on the respiratory manifestations, collecting gastro data on people with mild enteric symptoms or asymptomatic carriers might yield some interesting results.
Nervous system invastion
Previous research on SARS-CoV suggests the the novel coronavirus may also have neuroinvasive potential. SARS-CoV was detected in neurons in the brains of infected subjects, where it concentrated mainly in the brainstem. This 2002 and 2003 study of SARS patients showed SARS-CoV particles in the brain, almost exclusively localised in neurons. It is thought to have entered the brain through olfactory nerves or via a synapse-connections from the lungs and air passages. This aligns with some patients experiencing hyposmia (reduced ability to smell).
In experiments inoculating mice with a low dose of MERS-CoV, virus particles were later only detected in their brains and not their lungs.
SARS-CoV2 may be remaining latent in neurons which is why it is going undetected by the host immune system. Since the virus may employ this method of concealment, recovery from infection may not guarantee complete clearance from the body.
Genetic engineering of bat coronaviruses
An interesting paper I read was published in 2007 where scientists isolated SARS-CoV virus homologs from bats and then inserted an RNA sequence from SARS-CoV, endowing the bat viruses a new functionality – the ability to bind to human ACE2 receptors. Through gene editing by replacing an amino acid sequence between residues 323 and 505 with the corresponding sequence of SARS-CoV RBD (receptor binding domain) it was sufficient to drive ACE2 receptor usage.
This work proved the conserved nature of coronavirus sequences and the ease of which new genes and functionality can be inserted into related strains.
Dissolving claims of a more virulent L strain
A misleading analysis of the genetic data of 2019-nCoV by Tang et al. (2020) is circulating claims that 2 forms of the virus exist with one being more pathogenic than the other. An analysis by Oscar MacLean and team – Response to “On the origin and continuing evolution of SARS-CoV-2” – dispels this paper as misunderstanding and irresponsible to claim there are differences in transmission rates between strains.
So how are did they come to the conclusion that one strain was different to another? An analysis of several 2019-COV samples from around the globe showed the occurrence of 111 non synonymous mutations with a majority of samples showing one particular mutation which they deemed the L type. In their study they wrote:
“L type is derived from the S type with L (~70%) is more prevalent than S (~30%) among the sequenced SARS-CoV-2 genomes we examined. This pattern suggests that L has a higher transmission rate than the S type.”
MacLean argues that just because a certain mutation shows up more often in the samples analysed does not prove that strains containing that mutation are more pathogenic or have a higher rate of transmission.
The argument is that there are more factors at play here than a single mutation in the viruses sequence and there are many external factors at play. We need to take into consideration that transmission:
- Is a a random probabilistic event
- Rates of transmission by infected people depend on variety of reasons, for example if they use public transport, cough outwardly or fail to self-isolate
- The occurrence of Founder effect
The Founder effect is known to occur when a small seeder population of a species inhabits a new environment and the genetic information diversifies due to rapid mutations occurring in the population. When a small number of viruses enter a new population a large number of mutations will occur.
A phyogenetic tree of the virus can be seen at https://nextstrain.org/ncov
How to read a phylogenetic tree https://artic.network/how-to-read-a-tree.html
In the paper referenced above titled Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China, where the first 41 laboratory-confirmed cases infected with 2019-nCoV were analysed:
- The median age of patients was 49 years and 13 (32%) patients had an underlying disease.
- All 41 patients had pneumonia with abnormal findings on chest CT.
- 13 (32%) of patients were admitted to intensive care units, and 6 (15%) died.
- High concentrations of cytokines were recorded in plasma of critically ill patients infected with 2019-nCoV.
- Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]).
- Less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38).
- Dyspnoea (difficulty breathing) developed in 22 (55%) of 40 patients.
- The median time from illness onset to dyspnoea was 8 days.
- 26 (63%) of 41 patients had lymphopenia (abnormally low level of lymphocytes (white blood cells)).
- Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]).
- Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα.
I know a lot of us have been experiencing headaches and general tiredness from the stress and anxiety of unfolding circumstances out of our control. Since these are symptomatic of being COVID-19 positive it is further exacerbating our anxieties. From this study, although a small sample size of 41, we can see headaches were among the symptoms less prevalent and fever is almost guaranteed.
Of course there is still disparity here since a comparison of South Korea and Italy’s citizen testing showed that you can be asymptomatic yet still be COVID-19 positive, with the 20-29 year olds demographic having the highest quantity of positive tests while being asymptomatic.
Conclusive evidence on whether this non-symptomatic age group has been experiencing mild symptoms has yet to emerge.
Asymptomatic individuals as spreaders
The report covert coronavirus infections could be seeding new outbreaks presents some interesting statistics on the percentage of infected people showing no or mild symptoms may be responsible for transmitting the virus.
- On the Diamond Princess cruise ship, a floating incubator SARS-CoV-2 experiment of sorts, 18% of 700 infected individuals never showed symptoms.
- A Wuhan study estimated 37,400 (57%) were out and about with mild or no symptoms by the 18th February 2020.
- A Japanese study of their infected citizens evacuated from Wuhan reported that 4 (31%) out of 13 infected never developed symptoms.
These findings are either indicative of inaccuracies in the test yielding a number of false positives or the more disconcerting truth that many carriers of the virus are asymptomatic. Given the speed of spread around the globe and the pop-up of mysterious positive cases in our communities, the latter is highly probable.
Drug efficacy, vaccine and potential treatments
From the escalating moments of the outbreak, I’ve been garnering solace from an old professor, the Irish immunologist Luke O’Neill @laoneill111. Here are a couple of his updates:
Many drugs are being carefully tested against COVID19. 4 standouts optimism high: 2 that kill the virus- Remdesivir (anti-Ebola) and combo Ritonavir/Lopinavir (anti-HIV), and 2 anti-inflammatories to protect lungs (hydroxychloroquine (anti-malaria) and tocilizumab (anti-IL6).
3 companies Novartis Mylan and Teva agreed today to supply hydroxychloroquine tablets to fight COVID-19 for free. Big controlled trials running following prelim data. Novartis will provide 130 million, Mylan 50 million and Teva 16 million. Novartis plan for worldwide distribution
This is good news, especially after reading how the progress of Gilead’s remdesivir, a vaccine with proven efficacy against coronaviruses has been tied up by patent lawsuits.
A paper published by Hoffman et al. on March 5th 2020 confirmed that SARS-nCoV-2 uses the ACE2 receptor for binding and entry into the cell but also depends on TMPRSS2 a serine protease for S protein priming. This discovery may lead to important prevention and treatment of viral development through clinical use of a protease inhibitor.
Disrupting the mechanism of the S glycoprotein binding to host cells or the priming of the S glycoprotein yields great potential for disarming the spread and pathogenicity of the virus.
A more natural approach to prevent infection may be by consuming resveratrol, a natural compound found in grapes and red wine. A study showed resveratol was an effective antiviral against MERS-CoV in cultured cells. Resveratrol at varying concentrations was shown to reduce viral RNA reproduction and cell death. Resveratrol’s mechanism of viral inhibition in MERS-CoV could have efficacy against another family of coronavirus.
Despite the uncharted territory we are navigating, I have confidence in the scientific community to pull together and develop a vaccine or virus disrupting medication in the meantime. The stakes are high but there is a lot on our side – highly advanced knowledge of microbiology, genetics and bioinformatics techniques, open source software for viral sequence analysis Arctic Network and the ability to instantly share and communicate findings around the world enabling collaboration and education.
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