Technical notes on CoV2

• • • We should focus on the temperature-sensitivity of RNA secondary structure
    • The “s2m” structure in the 3’ untranslated region is of particular interest
    • The distribution of ACE2 may not be the main driver of viral tropism
    • Influenza has been shown to have a temperature-driven switch, with high temperature favoring transcription over replication

If scientists have followed my blog and read my paper, many of the experiments that we need to carry out should be obvious.  However, the following points may not be obvious:

1. We need to focus more on RNA secondary structure than protein sequences.  Microbiologists tend to focus on the surface proteins of viruses, if only because this is relatively easy to do.  I have in fact looked carefully at the sequences of influenza surface proteins from subpolar and tropical strains but saw no consistent trends.  In retrospect, this may not be surprising because changing an amino acid of a surface protein is quite a big step, and it doesn’t happen very often in influenza.  However we know that both influenza and coronavirus have highly conserved RNA secondary structure (and, in coronavirus, tertiary structure) in RNA that is not  translated.   This conservation means that the structures must be doing something.  Secondary structure is inherently temperature-sensitive, and many organisms (both microorganisms and higher organisms) make use of ‘‘RNA thermometers” [14].  These are RNA segments that respond to temperature changes with three-dimensional conformational changes that alter gene expression.  Because many individual bases contribute to each conserved RNA configuration, the virus has the potential to fine-tune its temperature-sensitivity by changing bases in these regions.  These regions might be good places to look for mutations that give rise to milder strains in influenza, coronavirus and other respiratory viruses.
2. s2m structure. In fact, coronavirus differs from most viruses in that it has well-defined RNA tertiary structure at the 3′ end of its single-stranded RNA genome.  This structure is called the s2m, and the s2m of CoV-2 is almost identical to that of the SARS virus.  It would be very interesting to know whether the s2m can act as an RNA thermometer.  Does it flip between two conformations somewhere between say 33 and 37°C?  The structure of SARS s2m was determined by Bill Scott’s lab at UC Santa Cruz.  See http://scottlab.ucsc.edu/2019-nCoV/ .
3. ACE2 surface-bound enzyme. There has been a lot of talk about the enzyme that is used by CoV-2 to gain entry into the cell.  This is called ACE2 and it’s common in the lungs, and it has been suggested that the distribution of ACE2 might explain the tendency for the virus to infect the lungs.  However a recent preprint by pointed out that there are as many ACE2-expressing cells in the nose and mouth as in the lungs and colon, so this seems not to be the whole story.  See https://www.researchsquare.com/article/rs-16992/v1
4. It’s interesting to look at the way influenza strains move around the world.  My suggestion predicts that viruses should move more often from tropical locations to temperate regions than in the opposite direction, because tropical strains are predicted to be less temperature-sensitive, and therefore more virulent.  The movement of influenza strains was investigated by Bedford and colleagues in 2015 [3].  Their data shows that 50% of European A/H3N2 influenza strains were descended from strains that were in tropical or subtropical regions one year earlier. For A/H1N1, B/Victoria-like and B/Yamagata-like influenza strains, the proportions were 62%, 17% and 32% respectively.  On a one-year timescale, H3N2, H1N1, and B/Yamagata/16/1988-like influenza were more likely to migrate from South to North China than in the opposite direction.
5. Several studies looked at the effect of temperature on RNA synthesis in influenza.  They found a “switch” that was controlled by temperature in both laboratory and recently-isolated strains.  High temperature favored transcription, while at low temperature replication was favored.  Strains investigated included WSN, A/PR/8/34, H3N2 Hong Kong flu, H1N1 swine flu, and two strains of H5N1 bird flu.  See pages 115-116 of my 2016 review in Medical Hypotheses.
6. Several respiratory viruses were found to be easier to isolate and replicate at 33 than 37.  Rhinoviruses were first isolated at 35°C but a greater variety of rhinoviruses was discovered at 33°C, and this is the temperature that is recommended today for their isolation by the Clinical and Laboratory Standards Institute. Coronaviruses were first isolated at 33°C.  Naturally occurring influenza strains are also frequently ts. For example, four viral H2N2 ‘‘Asian” influenza specimens isolated from patients 1962 were temperature-sensitive in tissue cultures and eggs.  In 1977 nine of ten ‘Russian” H1N1 influenza strains isolated in China by Kung were temperature-sensitive. Oxford et al. found several H1N1 and H3N2 isolates similar properties in 1980.  For other examples see page 115 of my review.
7. Several studies have reported viral dormancy and subsequent reactivation in Antarctic stations when they were completed isolated over the winter.  More recently, PCR studies have found asymptomatic infections that were followed a few weeks later by respiratory illness caused by the same or indistinguishable viruses.  See page 110 of my review.
8. Viruses sometimes establish “persistent infections” of cell cultures.  For this to happen, viruses generally need become less active in cells in order to establish a balance between cell and virus replication.  Several authors have noted that the resulting viral strains are often temperature-sensitive, growing faster at temperatures below the incubation temperature of the cell cultures.  Examples included Newcastle disease virus, Western equine encephalitis virus, Sendai virus, measles virus, vesicular stomatitis virus, Sindbis virus, mumps virus, and influenza.  See pages 114-115 of my review.
9.  The converse trend has been seen in at least two labs.  When influenza viruses were propagated at low temperature in conditions that allowed rapid growth, temperature-sensitivity was unexpectedly lost.  See page 115 of my review.
10. I have made suggestions in my review for experiments to test my hypothesis.  The problem could be tackled at many levels, from testing advice to individuals, to detailed lab and genetic work.  A high priority during the current epidemic is to test the temperature-sensitivity of CoV-2 at several points in its lifecycle, including binding to cells, entry into cells, movement through cells, transcription, replication of genetic material, assembly of virions and release from cells.

[1] Narberhaus et al. “RNA thermometers.” FEMS microbiology reviews 30.1 (2006): 3-16.

[2] Chursov et al. “Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus.” RNA biology 9.10 (2012): 1266-1274.

[3] Bedford, et al. “Global circulation patterns of seasonal influenza viruses vary with antigenic drift.” Nature 523.7559 (2015): 217-220.

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The Hypothesis

For a general discussion of the seasonality of respiratory viruses, written for the layperson, please see

Every winter, colds and flu increase

For detailed scientific information about the seasonality of respiratory viruses, including discussion of the trade-off model, viral dormancy and much else, see my 2016 paper:

Shaw Stewart, PD.  Seasonality and selective trends in viral acute respiratory tract infections. Medical Hypotheses 2016; 86 104–119.#

Also

Shaw Stewart PD, Bach JL. Temperature dependent viral tropism: understanding viral seasonality and pathogenicity as applied to the avoidance and treatment of endemic viral respiratory illnesses. Reviews in Medical Virology. 2021 May 3:e2241.  https://doi.org/10.1002/rmv.2241

For a discussion of the strange timing and duration of influenza epidemics, please see

The strange arrivals – and departures – of influenza epidemics in the UK, 1946-1974

Applications to Covid-19

For information about the probable seasonality of Covid-19, and whether we can expect it to become rarer in the summer, or reappear in the fall, please see

Predicting the seasonality of Covid-19

For comments about the epidemiology of Covid and other respiratory illnesses, please see

Epidemiology of respiratory illness

For discussion of how the trade-off model can be applied to the Covid epidemic see

Covid 19 and the trade-off model

For a simple model of the transmission of viruses such as CoV-2, please see

A simple model of CoV-2 transmission

For comments about how quickly we can expect viruses to adapt to new environments, please see

Adaptability or respiratory viruses

For practical tips on avoiding respiratory illness see

Suggestions for avoiding colds and flu – and Covid-19