SARS-CoV2 (Continued)

SARS-CoV2 (Continued)  

     One of the puzzling features of this virus is that some infected people, while carrying a considerable viral load, and shedding infectious virions, nevertheless develop such trivial symptoms that they never notice they are infected. While other, of course, develop virally driven hyper-inflammation,  respiratory failure, and sometimes also kidney and heart failure.

     This variability in response is especially striking when it affects a whole nation-state. Thus the official WHO figures record that Vietnam, with a population of 97 million, has confirmed only 416 cases of COVID-19, and that none have died. (c.f. UK, population 67 million, 297,914 cases, 45,677 deaths on 24th July. )

Possible explanations for variable responses.

     The hypothesis of genetic variability in the human host, which supposes that the Vietnamese lack e.g. the ACE2 receptor site (See my SARS-CoV2 post), is more-or-less ruled out by anecdotal observations such as that of an asymptomatic carrier infecting 5 family members [1]. As also is the hypothesis of genetic variability among the circulating SARS-CoV2 strains, for the carrier would obviously infect the household with the strain she was carrying. 

     Could there be competition between two co-infecting viral strains, where one causes trivial, often negligible, symptoms but occupies all the binding sites? 

     Or could there be, in some people, residual anti-bodies at a sufficient titre from a previous infection by the same (or sufficiently similar) coronavirus?  This last seems the best hypothesis, and in the last 10 days has received some support. 

    The group of Antonio Bertoletti at the Duke-NUS Medical School in Singapore has just published in Nature [2] an online report showing that previous infection with a virus of the beta-coronavirus family can leave long-lasting and multispecific T cell immunity to the nucleocapsid structural protein (N protein, or NP, See my Coronavirus post) that can cross-react with the N protein of SARS-CoV2). This previous infection could be a harmless "common cold" member of the corona virus family, but in Singapore it was possible also to study survivors of the 2003 SARS pandemic. 

     Back in 2013 a group in Taiwan explored the antigenicity of the N protein of the mild common cold virus HCoV-OC43, and had found that the middle section was highly antigenic [3]. Well over 90% of healthy young adults contained antibodies in their serum against the N protein of this common virus. These antibodies were even found in cord-blood samples showing that newborn babies acquire some immunity against coronaviruses from their mothers. 

     We have already learnt that it is foolish to infect yourself deliberately with SARS-CoV2; you could become very ill or die. But there may be a beta-coronavirus, prevalent in Vietnam, that does protect you against COVID-19. And it may be that here in Britain a sufficiently recent 'common cold' may leave you with enough circulating antibodies to prevent or greatly limit the effect of SARS-CoV2 infection. 

References

[1]  Susan Lee,  Paula Meyler,  et al. (2020) Can J Anaesth. : 1–7. "Asymptomatic carriage and transmission of SARS-CoV-2: What do we know?". 

[2]  Le Bert N, Tan AT, Kunasegaran K, et al. (2020)  "SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls." [published online ahead of print]. Nature. 2020;10.1038/s41586-020-2550-z. doi:10.1038/s41586-020-2550-z.  

See also  Ruairi J. Mackenzie, Science Writer for Technology Networks (2020) “'Common Cold' Coronaviruses Could Help Produce Anti-SARS-CoV-2 Immune Cells." 

[3]  Fang-Ying Liang, Leng-Chieh Lin, (2013  J Virol Methods;187(2):413-20. "Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: implications for the diagnosis of coronavirus infection. "

COVID is not retreating

Easing the Lock-down-2

(COVID-19 is no longer retreating)

     Using governmental data  for the "new UK cases each day", the numbers have been declining markedly since I started logging them on the 1st of May.  Then we were getting 6000 new cases per day, now only 500 - 700. 
     Now, if there are fewer active cases in the community there should be fewer new cases; i.e. the decline should be exponential, or logarithmic, like a cooling curve.  So, in the Figure below I have plotted the logarithm of the daily increment in UK cases against days (since the 1st of May, so 85 is the 24th July). (A value of 3.0 would mean 1000 new cases per day (1000=10^3); a value of 2.0 means 100 new cases per day (100=10^2).) 
    The data from 1st May to the 24th June are coloured blue, and fit a logarithmic decline. However, the data from 30th June to the 24th July, coloured red, show that the disease is no longer on the decline. We are in steady-state. 
     At the worst there could be a "second-wave", as we have been warned; at the best we shall live like this indefinitely, or until there is an effective and safe vaccine.

Semi-log plot of new cases per day in UK

(See text for details)

SARS-CoV2

SARS-CoV2

    After looking briefly into coronaviruses in general (see previous post), I have turned to SARS-CoV2, the virus responsible for the current pandemic of the respiratory infection called COVID-19.

Interesting aspect at this stage in the pandemic include:

• Important biochemical and biological differences between SARS-CoV2 and SARS-CoV viruses.
• Hight infectivity; 100 or 1000 times higher than SARS-CoV.
• Why are 30-40% of infected  'carriers' symptom-free?

Difference between SARS-CoV2 and SARS-CoV(1) that might cause higher infectivity.

    SARS-CoV2 is said to be 1000 time more infectious than SARS-CoV1; a pretty loose statement, but there is some biochemistry to investigate. Is this high infectivity due to:  

a.    different, more accessible or numerous, targets on host;

b.    tighter binding to target; 

c.     epidemiological factors like shedding before or without symptoms, or more coughing and sneezing; 

d.    better evasion of host responses. 

[a] Target on host

    The host receptor for both SARS-CoV and SARS-CoV2  seems to be the dimeric membrane-bound protease called ACE2 (for Angiotensin Converting Enzyme 2). There is a small mystery here, as the first investigators found very little ACE2 protein, or mRNA, in human lung tissue, though lots in arteries, gut, kidney, testes and elsewhere [1]. Yet SARS-CoV2  seems to attack the lower respiratory tract (as well as gut, blood-cells, kidney, etc). This was so important that the question was re-examined and some ACE2 was found in lung tissue, particularly around arterioles [2].  (It is notable, however, that SARS-CoV2  can cause diarrhoea and kidney damage [3].) Other coronaviruses act primarily as gut pathogens (See previous blog). I worried that the polyclonal antibody used by Hamming to test the presence of ACE2 (which was reared against a stretch of 19 amino acids distinctive to ACE2) might cross-react and mislead. However, it seems to be universally accepted that the receptor for SARS-CoV2  is ACE2.

[b] Tighter binding of Spike to Target.

    The spike protein is not highly conserved; the opposite rather, and it seems likely that mutations, deletions and insertions in spike can affect host range; possibly infectivity as well. There are distinctive features in the spike protein of SARS-CoV2 not found in SARS-CoV spike.  Thus, there is an insert of 4 amino acids (PRRA) into the sequence, which generates a cleavage site absent from the spike protein of SARS-CoV and several other coronaviruses (but present in MERS!). 
                                                                                ↓            
            SARS-CoV2: CASYQTQTNSPRRARSVASQSI
            SARS-CoV  : CASYQTQTNS­­– – – –RSVASQSI
Cleavage is effected by a host protease present cytoplasmically throughout the body. It is a 'subtilisin-type' calcium-dependent protease (called furin), which cleaves after the marked serine residue, but the cleavage site is flagged by the paired basic amino acids (–R+R+–). The furin enzyme is obviously present to service host proteins. But SARS-CoV2 is not unique among viruses in using it for pathologicial purposes, for furin also operates in the activation of: HIV, influenza, dengue fever, Marburg virus, papillomavirus, and even anthrax toxin. It is suggested that processing of progeny virions before release may facilitate the spread of virus (c.f. SARS-CoV)[4]. 
    The spike protein of SARS-CoV2 operates as two peptides (S1 and S2) formed into a trimeric "clove-like" structure.  Tai et al. (2020) were able to compare the binding (to human ACE2) of SARS-CoV2 spike with that of SARS-CoV spike, and found it bound 9 time more tightly. (Interestingly, it bound even more tightly to bat ACE2). [5] 

[c] Epidemiological factors.

     It is important to distinguish pre-symptomatic from truly a-symptomatic carriers; both categories of infected subjects experience no symptoms, but in the former case they eventually develop symptoms, while in the latter they never do. When I use the term 'asymtomatic' in this post it will always mean that the subject did not develop sysmptoms, in at least 4 weeks. Both categories can spread the disease.
    Compared with SARS-CoV of 2003, SARS-CoV2 causes more cases with mild (or very mild) symptoms, and larger numbers stayed at home in the community. There was also twice as long incubation period before the appearance of symptoms (4–12 days). Similarly relevant for the spread of the disease, the new strain can shed infective particles as soon as symptoms appear; or even before (see above). And they can continue shedding for 3 weeks [6].   Susan Lee et al. [7] mentions a family in Anyang (China) where an asymptomatic carrier tested positive for the virus and infected 5 family members. 
    Other factors of obvious relevance to infectivity are propensity to cough or sneeze. 

[d] Evasion of host defences.

    Two recent well referenced summaries are by Indwiani Astuti and Ysrafil [8] and Swatantra Kumar et al. [9]

 References

[1] Donoghue, M., Hsieh, F. et al. (2000) Circulation Research. 87:e1–e9; "A Novel Angiotensin-Converting Enzyme–Related Carboxypeptidase (ACE2) Converts Angiotensin I to Angiotensin 1-9".
[2] Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H.  (2004)"Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis." J Pathol.; 203:631-637. doi:10.1002/path.1570
[3] Martinez-Rojas, M.A. et al. (2020) "Is the kidney a target of SARS-CoV-2?"; Am J Physiol Renal Physiol.; 318:F1454-F1462.
[4] Coutard, B., Valle, C. et al  (2020)
Antiviral Res.; 176: 104742. "The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade."
[5] Tai, W-B. He L.,  Zhang, X-J. et.al. (2020) Cellular & Molecular Immunology volume 17, 613–620. "Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine"
[6] Petersen, E., Koopmans, M. et al (2020).Lancet, Infectious Diseases, https://doi.org/10.1016/ S1473-3099(20)30484-9 "Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics.".
[7] Lee S, Meyler P, Mozel M, Tauh T, Merchant R. "Asymptomatic carriage and transmission of SARS-CoV-2: What do we know?"  Can J Anaesth. 2020;1-7. doi:10.1007/s12630-020-01729-x
[8]  Indwiani Astuti & Ysrafil, (2020) Diabetes Metab Syndr. 2020 July-August; 14(4): 407–412.
Published online 2020 Apr 18. doi: 10.1016/j.dsx.2020.04.020 "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response"
[9]  Kumar S., Nyodu R., Maurya V.K., Saxena S.K. (2020) Host Immune Response and Immunobiology of Human SARS-CoV-2 Infection. In: Saxena S. (eds) Coronavirus Disease 2019 (COVID-19). Medical Virology: From Pathogenesis to Disease Control. Springer, Singapore. Published online 2020 Apr 30. doi: 10.1007/978-981-15-4814-7_5

Corona Viruses

Corona Viruses

(Some notes on Coronaviridae to put SARS-CoV2 into context)

Coronaviridae – the Corona Virus family

     The name “corona virus”, and the biological family name Coronaviridae, were coined in 1975 by Tyrrell and co-workers. However the viruses had been discovered ten years earlier, in 1965 by Tyrrell et al., and independently (in 1966) by Hamre & Procknow, in tissue or organ cultures inoculated with material from volunteers with “common colds” [1]. The salient features at this stage were [a] RNA viruses, [b] ether-sensitive (so with a lipoid envelope), [c] cultivable in cultured human tissue or organ cultures but not in fertilised chicken’s eggs, [d] isolated from volunteers with mild upper-respiratory tract infections [1].

     With the electron microscope it was possible to visualise particles of 80 to 150 nm diameter (c. 10^-4 mm), decorated with widely spaced club-shaped knobs, or ‘spikes’; this picture has become well-known the world over during the pandemic of SARS-CoV2 of 2019/2020 [1].  (It has been suggested that the name refers more to a solar corona than directly to a crown or coronet.)

    Considerable progress was made between 1965 and 2002 in understanding the biology of the these ubiquitous viruses that caused mild disease symptoms. Especially studied were two strains called OC43 and 229E. The continuous RNA strand is approximately 30,000 bases long, single-stranded and is in the ‘positive’ orientation (so it can be directly translated into protein, 5’-terminus of RNA corresponding to NH2-terminus of protein).  Like other RNA viruses, the genome is susceptible to frequent mutations. Immunity of the host tends to be transient, so an endemic strain of virus (like 229E in the USA) can cause wave after wave of epidemic disease in a population, recurring every 2-3 years [1].

Taxonomy and Evolution

     It is now believed that corona viruses are very ancient in evolutionary terms, having been around some 300 million years, i.e. as long as their bat and bird hosts [2]. Identification and taxonomy are both based on cloning up complimentary DNA using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), employing primers synthesised to match highly conserved parts of the genome; often in the RNA-dependent RNA polymerase (RdRP), coded at the overlap of the two large open reading frames (See Figure below). There are now held to be 4 genera of viruses in the family Coronaviridae (see Table). 

Table

Figure [See ref. 3]

    With the outbreak of Severe Acute Respiratory Syndrome (SARS) in China in 2003, interest in the Coronanviridae intensified. This was a dangerous disease with average fatality of those infected of around 10%, and much higher for the elderly. It spread readily from human to human by droplet infection, and indirectly by touching contaminated surfaces. It was found to be caused by a new coronavirus, named SARS-CoV. This strain is related to bat viruses, but seems to have jumped to a human from a palm civet [3]. Cases were eventually reported in 29 countries, mostly with links to east Asia. But the disease then died out. Apart from a small escape-infection from a Chinese laboratory in 2004 it has not been seen since, anywhere in the world. 

    In 2013 another novel coronavirus disease broke out in the Middle East with an even higher mortality rate of 35%. This Middle East Respiratory Syndrome (MERS) has remained confined to the Middle East, where it lingers still (some 14 cases were reported in the first 5 months of 2020). While SARS-CoV seems to have jumped from bat to man via civet cats, the MERS virus seems to have jumped via the dromedary camel, which may explain its restriction to Saudi Arabia and neighbouring countries.  It has been endemic in camels for at least 3 decades [3].

    Over the last 17 years, intense study of the SARS-CoV virus  (which for clarity I shall sometimes call SARS-CoV1) has laid the foundation of our knowledge of SARS CoV2, the closely related agent of our present COVID-19 pandemic. I shall therefore expand a bit on the biology of SARS-CoV1.

SARS CoV1 Molecular Biology

     This, like all coronaviruses, is an enveloped, RNA virus containing a single-stranded, positively orientated, RNA molecule of 29,751 nucleotides [4]. This RNA codes for 28 proteins: 4 Structural proteins (S=spike protein, E=envelope protein, M=membrane protein, N=nucleocapsid protein); 16 non-structural proteins, derived by cleavage from two large polyproteins; and 8 accessory proteins, so-called because they are non-essential in tissue culture, though presumably important in the wild. The 2 polyproteins are coded by Open Reading Frame 1a (pp1a) and Open Reading Frame 1b (pp1b). 

    The functions of S, M, and N are relatively straightforward; not so the multi-functional E protein:

● S, a glycoprotein expressed on the outer surface of the excreted virion, is the docking mechanism of the virus, and contains specificity for the host target site. In the case of SARS-CoV1 the target is human Angiotensin-Converting-Enzyme 2 (ACE2), which is primarily expressed in the lower respiratory tract. (The HCoV-229E S-protein is specific for the host protein CD13; the MERS-CoV spike binds to dipeptidyl piptidase, so target gut and kidney.) 

● M, the most abundant protein, and the one that defines the shape of the virion.

● N, a protein that binds the genomic RNA, (I suppose like the histones that wrap and protect our DNA). 

● E, the 'envelope' protein, contain a hydrophobic domain of 24-28 amino acids, so presumably spans the lipid membrane. It is the smallest of the structural proteins at 76 amino acids, binds to M in the virus membrane, where it may act as an ion-channel or 'viroporin' [5,6]. But a great fraction of the completed E peptides are not found in the membrane, and E seems to have several other functions. There is a PDZ-bininding-Motif (PBM) at the extreme carboxyl end of E, which suggests that it can bind to a PDZ motif on some host proteins. (Of the 320 such PDZ-containing proteins in the human, only 5 are known to bind the E protein of SARS-CoV1; (a) Na/K ATPase alpha-1 subunit, (b) stomatin, (c) syntenin, (d) PALS, and (e) BcL-xL. It is therefore easy to imagine that the virus can, by means of these protein-protein interactions, disrupt tight-junctions in the lung, and Na⁺ concentration in nerve and muscle, while (with syntenin) it could cause a 'cytokine storm'. [5] (See Pathology below.)

    Several of the non-structural proteins (nsp) are highly conserved. Thus, nsp1, coded at the extreme 5' end of the genome, is highly conserved between all coronaviruses, but has very little homology with anything else in protein and nucleotide data bases; it is unique to Coronaviridae. The protein, of about 20kD, may inhibit cell protein synthesis, perhaps by degrading messenger RNA.[7].  

    The RNA-dependent RNA polymerase (RdRP), coded at the junction betwee the two large open reading frames (see Figure) [8], is the enzyme that, with auxiliary proteins, replicates the genome, and creates smaller fragments of RNA that act as templates for protein synthesis [8]. RdRP is one of the most tightly conserved regions of the geneome and the primers used for diagnosis and taxonomy are usually based on sequence from this region. 

Pathology

A high proportions of cases of SARS-CoV1 (and especially of MERS) occurred among health care workers rather than close family members, from which it is can be inferred that shedding of infectious virions occurs well after the onset of symptoms and hospitalization. It was often found that, as symptoms of distress increase, viral load decreases. This suggested that part of the pathology is due to the immune response. (See also the 'cytokine storm' in Swine Flu.) A comparison between those that survive and those that succumb to severe infection points to a failure (in the latter) to switch from innate immunity to acquired immunity. [3] 

References

[1]  Kahn, Jeffrey S. &  McIntosh, K. (2005) The Pediatric Infectious Disease Journal: Vol 24, S223-S227, "History and Recent Advances in Coronavirus Discovery."

[2]  Wertheim J.O., Chu D.K.W., Peiris J.S.M., Pond S.L.K., Poon L.L.M. (2013) J Virol. 87, 7039–7045. "A case for the ancient origin of coronaviruses."

[3]  de Wit, E., van Doremalen, N., Falzarano, D., Vincent JM. (2016) Nat Rev Microbiol. ; 14: 523–534. 

[4]  Marco A., Marra, et al., (2003) Science, 300, 1399-1404

"The Genome Sequence of the SARS-Associated Coronavirus".

[5]  Wu, QingFa,  Zhang, YiLin, et al. (2003) Genomics, Proteomics & Bioinformatics, Volume 1, 131-144. "The E Protein Is a Multifunctional Membrane Protein of SARS-CoV".

[6]  Castaño-Rodriguez C, Honrubia JM, et al. (2018). mBio. 22;9(3):e02325-17.  "Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis."

[7]  Connor,  R.F. & Roper, R.L. (2007) Trends Microbiol; 15: 51–53. "Unique SARS-CoV protein nsp1: bioinformatics, biochemistry and potential effects on virulence."

[8]  Pasternak, A. O., Spaan, W.J.M., Snijder, E.J. (2006) J Gen Virol  87,1403–1421, "Nidovirus transcription: how to make sense…?"