The Cough Reflex and its Rôle in Virus Transmission.

The Cough Reflex and its Rôle in Virus Transmission.

(“Coughs and sneezes spread diseases.” Old folk-saying: )

Introduction

The cough reflex involves (a) triggering, or activation of a nociceptor, (b) an afferent signal in C-fibres of the vagus nerve to the brain, (c) efferent signals to the diaphragm and other muscles, and (d) a rapid muscular contraction [1]. 

We can all distinguish between a “dry” cough (when there seems nothing in the trachea or larynx to cough up), and a “wet” or “productive” cough (when phlegm or mucus is moved up by the blast of air travelling, we are told, at ≤ 600 mph.) It is the former that I am interested in. The brain has some (but limited) ability to suppress a cough, as also do placebo drugs [1]. But those who have struggled to suppress a cough during a concert, or when sharing a bed, know that it is extremely difficult. 

It has probably occurred to many others, as it has often occurred to me, that virus-infected mucous membranes generate a cough-trigger "in order to spread the virus itself" [2]. A virus that is able to trick its host into a cough (or a sneeze) could spread infectious virions widely, and thus increase enormously its chance of finding a new host. But the possibility that the corona viruses target only ACE2 precisely because ACE2 is crucial to the cough-trigger is an hypothesis too good to pass over. Is there a link between ACE2 and the cough reflex? Of all the cell-surface proteins to which the virus could bind, why does it  choose to bind to the ACE2? And does such binding trigger a cough? If so, how.

This led me to wonder what is the cough-trigger, and what part is played by an active viral infection. Dipping into the medical and scientific literature brought up three important areas for further study: Bradykinins, Angiotensins, and ACEs.

Bradykinins, Angiotensins, and ACEs

Bradykinin is a nona-peptide (which can be referred to as Bk(1-9)), but it can come with an additional N-terminal Lys residue (here called Bk(0-9)), or lose the C-terminal Arg becoming the more active Bk(1-8) (also called des-Arg9 Bk). Active bradykinins work via two G-protein-coupled receptors called B1 (especially linked to Bk(1-8) and strongly induced during inflammation), and B2 (activated by Bk(1-9) and constitutive, hence the ‘normal’ receptor).[3]

Angiotensins [4] are another small family of peptides, unrelated to the bradykinin sequences other than in size, charge, and having amino acid residues P and F in positions 7 and 8; see Table 1 below.) Angiotensin I is an inactive decapeptide (Ang(1-10)). It is converted to active (vasoconstrictive) angiotensin II (Ang(1-8)) by the removal of the c-terminal dipeptide. [It is probably irrelevant to this story, but angiotensin II raises blood pressure, while bradykinin(1-8) lowers it.]

ACEs, Angiotensin-Converting enzymes [5], are integral-membrane-bound proteolytic enzymes capable of cutting peptide chains. There are two related proteins; ACE1 and ACE2. The former converts inactive angiotensin I (the deca-peptide) to active angiotensin II (the octa-peptide), by cutting off the C-terminal dipeptide. [It also converts angiotensin(1-9) to angiotensin(1-7).]

ACE2 has become famous since January 2020 as the unique and highly specific binding site of the SARS-CoV-2 virus. It is present in a wide range of tissue surfaces, but especially in kidney, the endothelium of the gut and blood vessels, the lungs, and in the heart. It converts angiotensin(1-10) to angiotensin(1-9) by cutting off the C-terminal Lys. But it also degrades active Bk(1-8) to inactive Bk(1-7) and other peptides [6]. [It is a quaint irony of history that ACE1 cuts off a dipeptide while ACE2 cuts off a single amino acid residue.]

Table 1 Peptide Sequences (in one-letter code, showing net charge).

Bradykinins 

Bk(1-9)   RPPGFSPFR         ++ Active, vasodilator, B2

(Bk(0-9) LRPPGFSPFR         +++ ?B2)

Bk(1-8)   RPPGFSPF + Active, pain, B1

(Bk(0-8) LRPPGFSPF ++ ?B1)

Bk(1-7)   RPPGFSP         + Inactive,

Angiotensins

AngI(1-10) NRVYIHPFHL +++ Inactive

AngII(1-8) NRVYIHPF         ++ Active vasoconstrictor

Ang(1-9)       NRVYIHPFH +++ ?

Ang(1-7)       NRVYIHP         ++ Less active, competes AngII(1-8)

Is Bk(1-8) the Cough-Trigger?

There are three bits of evidence in favour of the hypothesis that the binding of virus to ACE2 might directly trigger cough by allowing the build up of Bk(1-8). 

    (1) A distinct and well documented type of “dry cough” is observed in 15-20% of patients taking ACE inhibitors to counter their high blood pressure [1]. These block the generation of active AngII(1-8), but also block the inactivation of active Bk1-8, which consequently builds up.Though not rigorously confirmed, it is widely assumed that this raised bradykinin level causes the cough. 

    (2) Raised bradykinin levels are found in virally infected mucosae [3]. 

    (3) Cough-Hypersensitivity-Syndrome, especially common in women [1,9] and in patients from south-east Asia [1], can be induced in animal models by local application of bradykinin. 

This hypothesis requires that virus binding (and subsequent inversion into the cell) blocks the action of ACE2 in deactivating Bk(1-8) to inactive Bk(1-7). The raised levels of Bk(1-8) after infection suggests that virus does have that effect.

References

[1]  https://www.sciencedirect.com/topics/medicine-and-dentistry/cough-reflex

[2]  Morice, A.H. Chronic cough hypersensitivity syndrome. Cough 9, 14 (2013). https://doi.org/10.1186/1745-9974-9-14

[3]  https://www.sciencedirect.com/topics/neuroscience/bradykinin

[4]  https://www.sciencedirect.com/topics/neuroscience/angiotensin

[5]  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321295/; https://www.frontiersin.org/articles/10.3389/fmed.2019.00136/full

[6]  https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(20)30282-6/fulltext; https://www.frontiersin.org/articles/10.3389/fmed.2019.00136/full

[7]  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340691/

[8]  https://www.sciencedirect.com/topics/neuroscience/bradykinin

[9]  https://erj.ersjournals.com/content/9/8/1624

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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. "

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…?"