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…?"