What makes SARS-CoV-2 variants more virulent?

Researchers from the Republic of Korea have conducted a study on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) – the agent that causes coronavirus disease 2019 (COVID-19) – that may help shed light on ‘Further studies on the increased virulence of variants of concern and interest.

The team – from the Korea Chemical Technology Research Institute in Daejeon – demonstrated that the four variants of concern currently in circulation exhibit differences in plate size, thermal stability, and replication rates.

Thermal stability correlated with plate size in all variants except line B.1.1.7 (alpha).

The largest average plaque size was observed for the B.1.351 (beta) variant, followed by P.1 (gamma), B.1.617.2 (delta) and alpha.

Young-Chan Kwon and colleagues suggest that the relatively large plaque size observed for beta may account for its increased thermal stability, which may, in turn, contribute to its increased transmissibility.

The researchers say these observations can help characterize the variants in terms of interactions with host factors and responses to environmental conditions.

Such studies will be crucial in informing effective management of the COVID-19 pandemic, they add.

A pre-printed version of the research paper is available on the site bioRxiv* server, while the article is subject to peer review.

Learn more about the worrisome variants currently in circulation.

Since the start of the COVID-19 pandemic at the end of December 2019, the genetic evolution of SARS-CoV-2 has led to the emergence of several new variants that are more virulent and infectious than the ancestral strain. Four of these variants (alpha, beta, gamma, and delta) have been classified as variants of concern by the World Health Organization.

These four variants share the D614G mutation which confers increased infectivity, possibly due to changes that affect the ability of the virus to bind and fuse with host cells. The N501Y mutation which is shared by alpha, beta and gamma is also known to increase binding affinity for host receptors and, therefore, cell entry.

However, the particular combination of mutations acquired by a variant could result in larger conformational changes and distinctive modifications, says Kwon and colleagues.

For example, variants of concern exhibit differential receptor binding affinity, with alpha requiring the most force to detach from host cell receptors, followed by beta, gamma, and delta.

What did the researchers do?

In the current study, the researchers found that each of the four worrisome variants exhibited differences in plaque size, the largest average size seen for beta, followed by gamma, delta, and alpha.

While there are many determinants of plaque size, the researchers hypothesized that differences in receptor binding affinity, thermal stability, and replication rate of these viruses were likely contributors.

The team assessed the thermal stability of the variants by measuring their infectivity and half-life after prolonged incubation (2, 4, 12 and 24 hours) at different temperatures. Infectivity was measured using a focus formation assay.

Of the four variants, beta exhibited the highest thermal stability, with a half-life approximately twice that of gamma or delta.

Correlation analyzes revealed a strong association between plate size and thermal stability in all variants except alpha. This variant had a relatively long half-life, despite a small plaque size.

The researchers say the results suggest that the relatively large plate size seen for the beta variant may account for its increased thermal resistance.

“This increased stability may contribute to its pathobiology and transmission, requiring further studies on viral titer and case fatality rate in humans,” they write.

Examination of viral replication rates of variants

Next, the team looked at the viral replication rates of the four variants in Vero E6 cells. A plaque formation assay was used to assess the number of infectious viral particles and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the concentration of viral RNA.

The alpha variant had fewer infectious viral particles than all other variants, and alpha and gamma had the lowest concentration of viral RNA.

The results suggest that although the alpha variant exhibits relatively high thermal stability, its rate of viral replication is likely low, thus contributing to its small plaque size.

Comparative analyzes of plate size, thermal stability and replication rate of four variants of SARSCoV-2.  (A) Representative images of the plaque formation test for each variant of concern.  (B) The average plate size (1E-3 mm2) of each variant.  (C) The relative infectivity of each variant after incubation at 4, 24 or 37 ° C for 8 h, assessed by a focus formation assay.  (D) Nonlinear regression of the relative infectivity of each variant after prolonged incubation (2, 4, 8, 12 and 24 h) at physiological temperatures (37 ° C).  (E) Mean half-life values ​​for each variant at 37 ° C. (F) The infectivity of progeny viruses was assessed by a plaque formation assay completed at 12, 24 and 48 h after infection. .  (G) Extracellular and (H) intracellular viral RNA was assessed by qRT-PCR with SARS-CoV-2 NP probes.  All error bars indicate standard errors of the mean.  Average plate size and half-life values ​​were compared using one-way analysis of variance in GraphPad Prism 8.0 software.  Statistical significance was set at p <0.05.

Comparative analyzes of plate size, thermal stability and replication rate of four variants of SARSCoV-2. (A) Representative images from the plaque formation test for each variant of concern. (B) The average plate size (1E-3 mm2) of each variant. (C) The relative infectivity of each variant after incubation at 4, 24 or 37 ° C for 8 h, assessed by a focus formation assay. (D) Nonlinear regression of the relative infectivity of each variant after prolonged incubation (2, 4, 8, 12 and 24 h) at physiological temperatures (37 ° C). (E) Mean half-life values ​​for each variant at 37 ° C. (F) The infectivity of progeny viruses was assessed by a plaque formation test completed at 12, 24 and 48 h after infection. . (G) Extracellular and (H) intracellular viral RNA was assessed by qRT-PCR with SARS-CoV-2 NP probes. All error bars indicate standard errors of the mean. Average plate size and half-life values ​​were compared using one-way analysis of variance in GraphPad Prism 8.0 software. Statistical significance was set at p

Further studies are needed

Kwon and colleagues say it will be important to analyze these worrisome variants in terms of both their clinical pathology and virology, as this will help improve understanding of their increased virulence.

“For example, the size of the plaque may be associated with contagiousness or viral transmission,” they write.

The researchers say more studies are needed to identify other determinants of plaque size, including functional mutations, interactions with host factors and environmental makeup.

“In addition, the variants of interest and other variants should also be studied to effectively control their spread,” the team concludes.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.

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About Hector Hedgepeth

Hector Hedgepeth

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