Exploration of the viral loads of the SARS-CoV-2 Omicron BA.5 and BA.2 sublines

In a recent study published on medRxiv* preprint server, researchers compared the viral loads of the Omicron BA.5 and BA.2 sublines of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

In January 2022, the number of patients infected with SARS-CoV-2 Omicron rapidly increased in Tokyo, Japan. After BA.2 replaced BA.1 as the predominant strain in April 2022, the number of patients gradually decreased. However, the infection rate of the BA.5 subline suddenly increased, and by July 2022, BA.5 had completely replaced BA.2. Although this phenomenon has been reported worldwide, its cause is still unknown. Therefore, it is important to examine the factors that make BA.5 more contagious than BA.1 and BA.2.

About the study

In the current study, researchers compared the number of virus copies corresponding to infection samples from patients with infections of the SARS-CoV-2 Omicron BA.1, BA.2 and BA.5 sublineage.

Polymerase chain reaction (PCR) tests were used to detect 774 patients with coronavirus disease 2019 (COVID-19) who visited Tokyo Medical and Dental University (TMDU) hospital between the February 1, 2021 and August 31, 2022. Nasopharyngeal swab specimens were collected during the patient’s first outpatient visit. In this investigation, the first sample was taken from each patient. Quantitative reverse-transcription PCR (RT-qPCR) was used to determine SARS-CoV-2 copy number in samples without ribonucleic acid (RNA) purification. Applying the threshold cycle numbers corresponding to each sample to the calibration line of reference samples of known concentration facilitated the calculation of the number of copies per 1 L of sample.

The melting curve of the PCR products was analyzed to identify the variations and types of sublines present. Additionally, the team extracted RNA from the swab samples. For E484K, L452R, N501Y and S371L/S373P, reverse transcription-PCR was performed with fluorescently labeled probes and primers. Moreover, the melting temperatures (T_m) estimated with melting curve analysis defined the types of variants and sublineages. A fifth of the samples were used for whole genome sequencing (WGS) to confirm subline types and variants identified by PCR.

Results

In the study, a total of 774 samples were examined. In PCR samples, the S371L/S373P probe was used to differentiate SARS-CoV-2 Omicron BA.1 and BA.2/BA.4/BA.5 and other Omicron variants. The team found that all of the samples that produced PCR results were Omicron variants. Samples from the test period did not contain any of the Alpha or Delta variants.

In particular, 215 samples had mutations 501Y, 484A, 452L and 371L/373P, called BA.1, while 184 samples had mutations 501Y, 484A, 452L and 371F/373P, called BA.2. Individuals belonging to the BA.2 cohort, six BA.2.12.1 samples with mutations 501Y, 484A, 452Q and 371F/373P, as well as ten unidentified BA.2 samples with mutations 501Y, 484A, 452M and 371F/373P .

Additionally, the PCR variant used in this investigation found 290 positive samples with 501Y, 484A, 452R and 371F/373P mutations and were classified as BA.5. Viruses of the BA.5 subline also possess the F486V mutation. Due to this mutation, the T_m of the E484K probe changes to 37°C, while the T_m BA.2 samples with 484A and 486F mutations remained constant at 40°C.

Genotyping results were similar to those resulting from PCR. Samples containing 501Y, 486V, 484A, 452R and 371F/373P were classified as BA.5 in subsequent analysis because WGS analysis did not detect the BA.4 sublineage in the samples. Additionally, BA2.75⁴ was also not detected by WGS analysis. Although the distribution of copy numbers for the BA.1, BA.2 and BA.5 sublines was widely dispersed, contrary to predictions, the copy numbers observed in the BA.5 cases were lower than those of the cases BA.2. Cases BA.5 and BA.1, as well as cases BA.2 and BA.1, did not differ significantly from each other.

Overall, the study results highlighted that compared to viral loads displayed by earlier variants and sublines, the increased infectivity of Omicron BA.5 is not primarily due to a higher viral load. To limit the spread of COVID-19, future research needs to investigate the specific mechanism underlying the high infectivity of SARS-CoV-2 variants.

*Important Notice

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