Mutations in novel coronavirus make it more dangerous

A new report published on the preprint server medRxiv in April 2020 reveals that the novel coronavirus SARS-CoV-2 shows the presence of new mutations acquired within infected human cases, which significantly increase its ability to cause human disease.

With the current novel coronavirus pandemic already having caused approximately 2.47 million infections and 170,000 fatalities, as of April 18, 2020, public attention is focused on what causes the relatively high mortality rate in this population. Currently, scientists have found a number of variant forms of the SARS-CoV-2 virus, which causes the COVID-19 pandemic.

The majority of infections are asymptomatic. The virus can spread during this asymptomatic period, making containment a real challenge, as shown by the current community spread of the illness in the US and many Western European countries. It also remains viable and capable of infection within aerosols for hours, and on surfaces for up to 7 days.

Study: Patient-derived mutations impact pathogenicity of SARS-CoV-2 Image Credit: ImageFlow / Shutterstock
Study: Patient-derived mutations impact pathogenicity of SARS-CoV-2 Image Credit: ImageFlow / Shutterstock

Why are mutations in the novel coronavirus important?

The virus gains entry to the host cell through the spike glycoprotein (S), which binds to the angiotensin-converting enzyme 2 (ACE2) molecule to enter the cell. This receptor molecule is found on a variety of tissues, including the epithelium of the nasal cavity, the lung, the spermatogonia, Leydig, Sertoli, and other gastrointestinal epithelium.

Seven betacoronaviruses cause disease in humans, in varying severity. The most variable part of the betacoronavirus genome is the part encoding the S protein’s receptor-binding domain (RBD). Multiple variations have been detected within the SARS-CoV-2 genome as well, with many of them comprising the addition, replacement, or deletion of single nucleotides – these are called single nucleotide variants (SNVs).

The current study deals with the question: are these mutations important as far as the pathogenicity of the virus is concerned? In other words, do any of these SNVs make the viral illness more or less severe, infectious, or both? The answer could decide how vaccines are developed as well as new drugs to counter this disease.

How was the study done?

The researchers sequenced the genomes of 11 viral isolates of the SARS-CoV-2 from a Chinese hospital in Hangzhou, China. This is situated over 750 kilometers to the east of Wuhan, where the outbreak originated.

The 11 patient samples were collected in the first days of the Wuhan outbreak, between January 22 and February 4, 2020. Ten persons had either worked in, traveled to, or had close contact with inhabitants of Wuhan. The last was a contact of a confirmed COVID-19 case.

The patients had moderate or severe symptoms, three had underlying medical, and one required intensive medical care. All recovered from the illness.

The Novaseq 6000 platform was used to accomplish the super-deep sequencing of the 11 isolates. This showed the presence of 1-5 mutations in the coding sequences of the isolates. There were also mixed viral populations.

The researchers mixed the 11 isolates with cultured cells and then evaluated the viral load at 1, 2, 4, 8, 24, and 48 hours later, as well as cytopathic effects (CPE) at 48 and 72 hours later.

What did the study show about the mutations?

The ultra-deep sequencing showed 33 mutations. Of these, ten were in mixed populations. Nineteen were seen for the first time when compared against the library of over 1100 mutations stored at GISAID. Several of these were founder mutations; that is, each can be traced back to one original virus that generated a large group of viruses. These groups are now found in patients around the world.

One mutation was previously seen only in Australia but was observed in 2 isolates from patients who attended a conference with patients from Wuhan. A third attendee showed a novel mutation, related to the other one but at a different position of the same codon.

Another showed three consecutive mutations in a single gene, introducing two mutations at the level of the protein, as well as a fourth non-consecutive one. Overall, the analysis of a small number of isolates resulted in highly diverse mutations.

Two of the mutations produced the same mutations at different locations on the same codon. One was observed in 5 isolates, suggesting its presence early in the pandemic and in many residents of Wuhan, but is not significantly present in the GISAID data available at the time of the article’s publication.

There was a 3-nucleotide mutation in one patient, which created a highly potent viral strain, in terms of both viral load and CPE. This patient remained positive for the virus for 45 days.

The researchers also did an in vitro assay to assess the effect of the mutations on the isolates. All the isolates were mixed with cultured cells, and the results were observed at 1, 2, 4, 8, 24, and 48 hours later. The CPE was also observed at 48 and 72 hours from infection.

To detect the SARS-CoV-2 virus directly, they carried out specific rtPCR directed against three viral genes - the ORF1a, E, and N genes. All three groups of rtPCR showed very similar results. The cycle threshold was used as a measure of viral load. The lower the Ct, the higher the load.

How did mutations affect pathogenicity?

The investigators reported that all samples showed no evidence of viral replication at 1, 2, and 4 hours from infection. Viral load began to increase at 8 hours, for a few strains, and 9/11 strains showed high viral load by 24 hours. Some strains showed a much faster rate of increase than others.

At 48 hours, the viral load curve flattened out, except for two which had already reached a plateau earlier, at 24 hours. The viral load of strains at the two ends of the spectrum differed by almost 270 times. This leads to statistically significant variations between the strains producing the greatest and least viral load at 48 hours.

In other words, different viral isolates carrying different mutations have a significantly different viral load. The cytopathic effect or cell death rates are proportional to the viral load.

How is the study important?

The researchers say the high incidence of different kinds of mutations in a relatively small sample is due to the early timing of sampling within a limited geographical area of the index case in Wuhan. Some of the mutations do provide obvious benefits or disadvantages under specific circumstances. For instance, some mutations are found very commonly in the S protein-ACE2 protein interface.

There was a marked difference in viral load at 48 hours in the cells infected by different viral strains, showing that different mutations do change the replicative and disease-causing potential of the virus.

The study shows how the genotype affects the phenotype in most cases, with the in vitro effect promising to reveal how different viral strains behave as they acquire different mutations. Finally, the researchers note that 3 of the isolates were from stool samples, in contradistinction to another report indicating that a viable virus could not be isolated from such samples. This shows the viral capacity to replicate in these samples too.

The study concludes that “viral surveillance should also be performed at the cellular level when possible” since the mutations occurring within patient populations do affect the pathogenicity of the virus. Another suggestion is the identification of all founder mutations in the larger clusters of viruses circulating in the world at present, to find useful differences in pathogenicity. The development of vaccines and drugs will also hinge on whether these mutations affect therapeutic efficacy.

Important Notice

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

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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