Similar viral loads in Omicron infections regardless of vaccination status

BackgroundAlthough SARS-CoV-2 booster vaccinations are underway, breakthrough infections with Omicron variants are occurring. This study analyzed associations between Omicron sublineage (BA.1.1 and BA.2) viral load and vaccination history. MethodsViral loads in nasopharyngeal swabs were evaluated by quantitative real-time PCR, and the virus strain was evaluated by whole-genome analysis or TaqMan assay. ResultsA total of 611 patients positive for an Omicron SARS-CoV-2 variant were included; 199 were unvaccinated, 370 had received two vaccine doses, and 42 had received three doses. Similar viral loads and Ct values of BA.1.1 and BA.2 were detected regardless of vaccination history. No correlations between age and BA.1.1 and BA.2 viral load were observed. ConclusionOmicron-infected patients who had received a third vaccine dose had viral loads similar to patients with two doses or who were unvaccinated.

SARS-CoV-2 Omicron sublineage BA.2 replaces BA.1.1: genomic surveillance in Japan from September 2021 to March 2022

ObjectiveThe new emerging Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently spreading worldwide. We aimed to analyze the genomic evolution of the shifting Omicron virus subtypes. MethodsThe study included 1,297 individuals diagnosed as SARS-CoV-2 positive by PCR test or antigen quantification test from September 2021 to March 2022. Samples were analyzed by whole genome sequencing analysis (n=489) or TaqMan assay (n=808). ResultsAfter the outbreak of the SARS-CoV-2 Delta strain, the Omicron strain spread rapidly in Yamanashi, Japan. BA.1.1 was the predominant sublineage of the Omicron strain from January to mid-February 2022, but the number of cases of sublineage BA.2 began to increase after mid-February, and this sublineage was shown to have replaced BA.1.1 by the end of March 2022. We observed higher viral and antigen levels of sublineage BA.2 than of sublineage BA.1.1 in nasopharyngeal swab samples. However, no difference in viral load by patient age was apparent between sublineages BA.1.1 and BA.2. ConclusionsA transition from sublineage BA.1.1 to sublineage BA.2 was clearly observed over approximately one month. Omicron sublineage BA.2 was found to be more transmissible owing to its higher viral load regardless of patient age.

Classification of Omicron BA.1, BA.1.1 and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data

ObjectiveRecently, the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread and replaced the previously dominant Delta strain. Several Omicron sublineages (BA.1, BA.1.1 and BA.2) have been identified, with in vitro and preclinical reports showing that the pathogenicity and therapeutic efficacy differs between BA.1 and BA.2. We sought to develop a TaqMan assay to identify these subvariants. MethodsA TaqMan assay was constructed for rapid identification and genotyping of Omicron sublineages. We analyzed three characteristic mutations of the spike gene, {Delta}69-70, G339D and Q493R, by TaqMan assay. The accuracy of the TaqMan assay was examined by comparing its results with the results of whole genome sequencing (WGS) analysis. ResultsA total of 169 SARS-CoV-2 positive samples were analyzed by WGS and TaqMan assay. The 127 samples determined as BA.1/BA.1.1 by WGS were all positive for {Delta}69-70, G339D and Q493R by TaqMan assay. Forty-two samples determined as BA.2 by WGS were negative for {Delta}69-70 but positive for G339D and Q493R by TaqMan. The concordance rate between WGS and the TaqMan assay was 100% (169/169). ConclusionTaqMan assays targeting characteristic mutations are useful for identification and discrimination of Omicron sublineages.

SARS-CoV-2 B.1.1.7 lineage rapidly spreads and overwhelms R.1 lineage in Japan: serial and stationary observation in a community

BackgroundThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulates in the world and acquires mutations during evolution. To identify the new emergent variants, the surveillance of the variants of concern (VOC) and variants of interest (VOI) is ongoing. This study aimed to determine how the transition of viral lineage occurred by stationary genome analysis in Yamanashi, Japan. MethodsWe performed the whole genome sequencing using SARS-CoV-2 positive samples (n=325) collected from February 2020 to the end of June 2021. The number of analyzed samples accounted for 15.4% of the total 2,109 samples identified in our community. Viral lineage was defined by the Phylogenetic Assignment of Named Global Outbreak (PANGO) lineages. ResultsWe identified 13 types of viral lineages including R.1, P.1, B.1.1.7 (Alpha) and B.1.617.2 (Delta) These virus lineages had distinct periods of expansion and decline. After the emerging of the R.1 lineage harboring E484K variant (designated VOI in Japan), the prevalent B.1.1.214 lineage were no longer identified. The R.1 lineages were temporarily prevalent afterwards, but the influx of B.1.1.7 lineage (designated VOC) led to a decline in R.1. Currently, B.1.1.7 has become dominant after mid-April, 2021. ConclusionWe clearly elucidated the transition and replacement of viral lineage by the community-based analysis. The virus completely replaced by more infectious lineages, therefore, it will be necessary to continue to monitor the VOC and VOI.

Household transmission of SARS-CoV-2 R.1 lineage with spike E484K mutation in Japan

We aimed to investigate SARS-CoV-2 emerging lineage harboring variants in receptor binding domain (RBD) of spike protein in Japan. Total nucleic acids were subjected to whole genome sequencing on samples from 133 patients with coronavirus disease (COVID-19). We obtained the SARS-CoV-2 genome sequence from these patients and examined variants in RBD. As a result, three patients were infected with SARS-CoV-2 harboring E484K mutation in January 2021. These three patients were relatives; one was in the 40s, and two were younger than 10 years old. They had no history of staying abroad and were living in Japan. This strains were classified into GR clade (GISAID), 20B clade (Nextstrain) and R.1 lineage (PANGO). As of March 5, 2021, the R.1 lineage have been identified in 305 samples and dominantly observed in the USA (44%, 135 / 305) and Japan (28%, 84 / 305) from the GISAID database. During the period between October 26, 2020 and February 23, 2021, the frequency of the R.1 lineage was 0.97% (84 / 8,629) of the total confirmed data in Japan and 0.15% (135 / 90,450) in the USA. Although SARS-CoV-2 R.1 lineage was not globally predominant as of March 2021, further analysis is needed to determine whether R.1 variant will disappear or expand in the future.

Discovery of SARS-CoV-2 strain of P.1 lineage harboring K417T/ E484K / N501Y by whole genome sequencing in the city, Japan

On the February 2020, the very first case was an American female from Diamond Princess cruise ship. Since, we have confirmed 136 patients infected with coronavirus disease 2019 (COVID-19) until February 2021. Here, we conducted the whole genome sequencing analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on samples from 70 of 136 patients (51.5%). These patients were infected in Diamond Princess cruise ship (n=1), Africa (n=2), Japan (n=66) and Brazil (n=1). The viral genome sequence of a patient on the Diamond Princess cruise ship in February 2020 was similar to that of original strain found in Wuhan, China (19A clade). Four patients, including two returnees from Africa and two lived in Japan, confirmed at the end of March 2020 had sequences similar to those of lineage with D614G mutation, which was endemic in Europe (20A [n=3] and 20B [n=1] clade). The 64 Japanese patients confirmed from September 2020 to January 2021 had sequences similar to those of the currently prevalent lineage (20B [n=58] and 20C clade [n=6]). Subsequent analysis revealed three mutations (K417T/ E484K / N501Y) in the receptor binding domain of the spike protein in a man in his 40s. The sequence was identical to the P.1 lineage (also known as 20J/501Y.V3) reported in Brazil. This is the first report of SARS-CoV-2 P.1 lineage identified in the city, Japan.

Pooling RT-PCR test of SARS-CoV-2 for large cohort of 'healthy' and infection-suspected patients: A prospective and consecutive study on 1,000 individuals

BackgroundSARS-CoV-2 testing reagents are expected to become in short supply worldwide. However, little is unknown whether the pooling strategy detects SARS-CoV-2 with accuracy. MethodTo validate the feasibility of pooling samples, serial dilution analysis and spike-in experiment were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2 positive and negative patients. Furthermore, we studied a total of 1,000 individuals, who were 667 "healthy" (195 healthcare workers and 472 hospitalized patients with other disorders than COVID-19 infection) individuals and 333 infection-suspected patients with cough and fever, were tested. ResultsSerial dilution analysis showed the limit of detection of around 10-100 copies according to National Institute of Infectious Diseases, Japan. Spike-in experiment demonstrated RT-qPCR detect positive signal in pooling samples of SARS-CoV-2 negative and positive patient at the 5-, 10-, 20-fold dilution. By screening with pooling strategy by the end of April, 2020, there are 12 COVID-19 patients in 333 infection suspected patients (3.6%) and zero in 667 "healthy". We obtained these results with total running 538 times (instead of 1,000 times) by pooling strategy. ConclusionPooling samples is feasible for saving test reagents and detecting SARS-CoV-2 in clinical setting to prevent the spread of the virus and nosocomial transmission.

Analysis of Covid-19 and non-Covid-19 viruses including influenza viruses to see the influence of intensive preventive measures among Japanese

ABSTRUSTSevere acute respiratory coronavirus 2 (SARS-CoV-2) spread and cause death in worldwide. The preventative measures and infection control are underway throughout the society and there are signs of convergence in some areas. Other viruses as well as SARS-CoV-2 cause cold-like symptoms and spread in winter. However, it is unclear to what extent SARS-CoV-2, influenza virus and other causative viruses have been prevailed since the preventative measures were implemented. In this study, we conducted multiples PCR and quantitative reverse transcription PCR using nasal swabs from 191 patients with cold-like symptoms in Japan to reveal the causative viruses. As a result, at least one virus were detected in 40 out of 191 (21%) patients. Of these, we frequently identified the human rhinovirus / enterovirus (5.8%, n=11), SARS-CoV-2 (4.2%, n=8) and human metapneumovirus (3.7%, n=7). On the other hand, no influenza virus was detected. These results shows the prevalence of causative viruses after the social preventative measures and implies the difference of infectivity between SARS-CoV-2 and influenza virus.

Double-Quencher Probes Improved the Detection Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by One-Step RT-PCR

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges in Wuhan City, Hubei Province, spreads worldwide, and threats the human life. The detection of SARS-CoV-2 is important for the prevention of the outbreak and management of patients. Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay detected the virus in clinical laboratory. MethodsThis study utilized primers and single-quencher probes in accordance with the Centers for Disease Control and Prevention (CDC) in the USA and the National Institute of Infectious Diseases (NIID) in Japan. Moreover, we designed the double-quencher probes (YCH assay) according to the oligonucleotide sequence established by NIID. Using these assays, we conducted a one-step real-time RT-PCR with serial DNA positive control to assess the detection sensitivity. ResultsThe threshold cycle (Ct) value of RT-PCR was relatively low in CDC and YCH assays compared to NIID assay. Serial dilution assay showed that both CDC and YCH assays could detect a low-copy number of DNA positive control. The background fluorescent signal at the baseline was lower in YCH than that of NIID. ConclusionDouble-quencher probes decreased background fluorescent signal and improved detection sensitivity of SARS-CoV-2.