key: cord-324480-7u5lh4jx
authors: Sharma, A.; Preece, B.; Swann, H; Fan, X.; McKenney, R.J.; Ori-McKenney, K.M.; Saffarian, S.; Vershinin, M.D.
title: Structural stability of SARS-CoV-2 degrades with temperature
date: 2020-10-14
journal: bioRxiv
DOI: 10.1101/2020.10.12.336818
sha: 
doc_id: 324480
cord_uid: 7u5lh4jx

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter. Statement of Scientific Significance The economic and public health impact of the COVID-19 pandemic are very significant. However scientific information needed to underpin policy decisions are limited partly due to novelty of the SARS-CoV-2 pathogen. There is therefore an urgent need for mechanistic studies of both COVID-19 disease and the SARS-CoV-2 virus. We show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. Our nanoscale results are consistent with recent observations at larger scales. Our work strengthens the case for COVID-19 resurgence in winter.

SARS-COV-2 is a virus of zoonotic origin which was first identified in humans in late 2019 [1] . Similar to other coronaviridae [2] , the viral particles are enveloped and polymorphic decorated by a variable number of S protein spikes on their membrane [3] . One of the most confusing and yet urgently pressing questions at the time of this writing is whether the COVID-19 pandemic caused by SARS-CoV-2 will show seasonal character. Climate and seasonal dependence was expected early in the pandemic [4] due to similarity with other human coronavirus diseases [5] , however the rates of infections have failed to strongly decline in the summer of 2020, leading to widespread doubts about COVID-19 seasonality. At the same time, a mounting body of evidence, from theoretical studies [6] to experimental research on viral populations and their infectivity [7, 8] suggest that seasonality is indeed to be expected. However an understanding of how SARS-CoV-2 survives different environmental conditions is still incomplete and mechanisms of virus particle degradation are poorly mapped out.

This then creates uncertainty for public health policy and its forward projection.

A key challenge in studying SARS-CoV-2 is the extreme level of threat associated with the live virus and the resultant need for high safety standards for such work. Aside from the envelope and S proteins, SARS-CoV-2 also packages the positive sense RNA genome encapsidated with thousands of copies of nucleocapsid, N proteins. SARS-CoV-2 also packages thousands of copies of matrix protein (M) which consists of three membrane spanning helixes with small intraluminal and extra luminal domains. In addition, an unknown number of envelope (E) proteins, which contain a single membrane spanning helix, are also packaged in each virion.

We have previously shown that similar to SARS-CoV [9] , the expression of SARS-CoV-2 M, E, and S proteins in transfected human cells is sufficient for the formation and release of virus like particles (VLPs) through the same biological pathway as used by the fully infectious virus [10] . These VLPs faithfully mimic the external structure of the SARS-CoV-2 virus. The VLPs however, possess no genome and thus present no infectious threat which enables rapid studies with reduced safety requirements. The ability to produce non-infectious VLPs further enabled us to devise and rapidly validate novel strategies for manipulation of these particles, most notably via the addition of protein tags to the S and M proteins (these findings are detailed in a separate manuscript).

Here, we report studies of VLPs subjected to variable temperature conditions before or after being immobilized and dried out on a functionalized glass surface. We show that exposure of VLPs to a mildly elevated temperature (34 C) for as little as 30 minutes is sufficient to induce structural degradation. The effect is weaker for particles exposed to elevated temperatures in solution and stronger for exposure in the dry state. Overall, these results provide insight into the viral seasonality of SARS-CoV-2.

During initial refinement of VLP purification strategies and associated VLP characterization [10] , we have found that such particles remain stable for at least a week if stored in liquid buffer at near 0 °C conditions (on ice). We therefore examined whether they would remain stable at room temperature under dry conditions on a model surface (Fig. 1 ). Spytag-S VLPs were adhered to microtubules (MTs) are harder to image stably due to high amount of background noise which obscures poly-L-lysine inhomogeneity and MT washout sites (likely due to loose debris on the surface -plausibly a byproduct of particle degradations). Features consistent with intact VLPs are prominent relative to noise levels and hence easy to resolve (Fig. 2 ), but they are so exceedingly rare at 34 °C that they are considered outliers (Fig. 3 ). They were seen in large area surveys in which each particle is mechanically probed only a few times. Such particles do not survive intact during even a single detailed zoomed-in scan (Fig. 2) . It takes 20-30 minutes to install the sample into the AFM, approach the surface and validate tip condition. Therefore mildly elevated temperature has a rapid effect on VLP integrity. VLPs incubated at 34 °C in solution and imaged at room temperature ( Fig. 1C ) survive better than particles at 34 °C under dry conditions but still often appear disrupted or aggregated in AFM imaging.

A common transmission route for SARS-CoV-2 is through bioaerosols created during sharp exhalation events such as sneezing or coughing. The bioaerosol droplets tend to dry out quickly due to high surface area and small volume [11, 12] . Therefore virus particles may be exposed to both wet and dry conditions before coming into contact with and infecting the next host. It is widely recognized that virus particles often spread after their deposition on various surfaces [13] (although direct contact of the next host with contaminated bioaerosol may also be a viable route) and it is therefore also appreciated that virus particles can survive on various surfaces for an extended length of time [14] .

The ability to make VLPs based on the SARS-CoV-2 genome, combined with abundant available structural information allowing for high precision design strategies for the VLPs, opens a unique opportunity for fast progress and allowed us to overcome the safety concerns associated with experiments on the full virus. Here we used this technology to study the stability of the viral envelope and associated proteins (M, E, and S) under different environmental conditions. As might be expected a priori, the VLPs do indeed degrade when exposed to elevated temperatures. Our AFM imaging revealed that negligibly few particles retain their shape and even those exceptional particles degraded nearly instantly during scanning and hence are likely already structurally impaired. The MT washout sites are readily identifiable for scans at room temperature, but are difficult to see with an identical colormap presentation due to elevated background noise. However, artificially extending the z range of the data helps reveal the presence of MT washout sites (right panel, washout site highlighted with red oval). Faint modulation in the rightmost image is most likely due to electronic noise in the imaging system although a mechanical vibration noise contribution cannot be excluded.

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