Identification of Various Recombinants in a Patient Coinfected With the Different SARS-CoV-2 Variants.

BACKGROUND: Viral recombination that occurs by exchanging genetic materials between two viral genomes coinfecting the same host cells is associated with the emergence of new viruses with different virulence. Herein, we detected a patient coinfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants and identified various recombinants in the SARS-CoV-2 full-length spike gene using long-read and Sanger sequencing. METHODS: Samples from five patients in Japan with household transmission of coronavirus disease 2019 (COVID-19) were analyzed using molecular assays for detection and identification of SARS-CoV-2. Whole-genome sequencing was conducted using multiplex PCR with short-read sequencing. RESULTS: Among the five SARS-CoV-2-positive patients, the mutation-specific assay identified the Delta variant in three, the Omicron variant in one, and an undetermined in one. The undermined patient was identified as Delta using whole-genome sequencing, but samples showed a mixed population of Delta and Omicron variants. This patient was analyzed for viral quasispecies by long-read and Sanger sequencing using a full-length spike gene amplicon. In addition to the Delta and Omicron sequences, the viral quasispecies analysis identified nine different genetic recombinant sequences with various breakpoints between Delta and Omicron sequences. The nine detected recombinant sequences in the spike gene showed over 99% identity with viruses that were detected during the Delta and Omicron cocirculation period from the United States and Europe. CONCLUSIONS: This study demonstrates that patients coinfected with different SARS-CoV-2 variants can generate various viral recombinants and that various recombinant viruses may be produced during the cocirculation of different variants.

Prolonged shedding of infectious viruses with haplotype switches of SARS-CoV-2 in an immunocompromised patient.

A concern has been raised that the persistent COVID-19 infection in an immunocompromised host can be the source of the SARS-CoV-2 variants. This is the case of a 61-year-old man in complete remission of a follicular lymphoma after six cycles of rituximab and bendamustine with additional two cycles of rituximab completed eight months prior to the episode of COVID-19 pneumonia. The patient's respiratory failure was long-lasting, and required mechanical ventilation until day 75. Acquired immunity tested negative throughout the observational period. The viral RNA was detectable until day 100 while the infectious virus was isolated until day 79. Seven haplotypes were identified and the non-synonymous mutations accumulated in the spike gene which included E484Q and S494P. In the management of COVID-19 cases with suppressed immune statuses, initial evaluation of existing immunity and monitoring for infectiousness throughout the clinical course including the convalescent stage may be necessary.

Effect of High Pressure Processing on a Wide Variety of Human Noroviruses Naturally Present in Aqua-Cultured Japanese Oysters.

The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.

Effect of High-Pressure Processing on Human Noroviruses in Laboratory-Contaminated Oysters by Bio-Accumulation.

The contamination of oysters with human noroviruses poses a human health risk, since oysters are often consumed raw. In this study, human norovirus genogroup II was allowed to bio-accumulate in oysters, and then the effect of high-pressure processing (HPP) on human noroviruses in oysters was determined through a polymerase chain reaction (PCR)-based method with enzymatic pretreatment to distinguish infectious noroviruses. As a result, oysters could be artificially contaminated to a detectable level of norovirus genome by the reverse transcription-PCR. Concentrations of norovirus genome in laboratory-contaminated oysters were log normally distributed, as determined by the real-time PCR, suggesting that artificial contamination by bio-accumulation was successful. In two independent HPP trials, a 1.87 log10 and 1.99 log10 reduction of norovirus GII.17 genome concentration was observed after HPP at 400 MPa for 5 min at 25°C. These data suggest that HPP is a promising process of inactivation of infectious human noroviruses in oysters. To our knowledge, this is the first report to investigate the effect of HPP on laboratory-contaminated noroviruses in oysters.

The Relation between Hepatotoxicity and the Total Coumarin Intake from Traditional Japanese Medicines Containing Cinnamon Bark.

Cinnamon bark is commonly used in traditional Japanese herbal medicines (Kampo medicines). The coumarin contained in cinnamon is known to be hepatotoxic, and a tolerable daily intake (TDI) of 0.1 mg/kg/day, has been quantified and used in Europe to insure safety. Risk assessments for hepatotoxicity by the cinnamon contained in foods have been reported. However, no such assessment of cinnamon bark has been reported and the coumarin content of Kampo medicines derived from cinnamon bark is not yet known. To assess the risk for hepatotoxicity by Kampo medicines, we evaluated the daily coumarin intake of patients who were prescribed Kampo medicines and investigated the relation between hepatotoxicity and the coumarin intake. The clinical data of 129 outpatients (18 male and 111 female, median age 58 years) who had been prescribed keishibukuryogankayokuinin (TJ-125) between April 2008 and March 2013 was retrospectively investigated. Concurrent Kampo medicines and liver function were also surveyed. In addition to TJ-125, the patients took some of the other 32 Kampo preparations and 22 decoctions that include cinnamon bark. The coumarin content of these Kampo medicines was determined by high performance liquid chromatography (HPLC). TJ-125 had the highest daily content of coumarin (5.63 mg/day), calculated from the daily cinnamon bark dosage reported in the information leaflet inserted in each package of Kampo medicine. The coumarin content in 1g cinnamon bark decoction was 3.0 mg. The daily coumarin intake of the patients was 0.113 (0.049-0.541) mg/kg/day, with 98 patients (76.0%) exceeding the TDI. Twenty-three patients had an abnormal change in liver function test value, but no significant difference was found in the incidence of abnormal change between the group consuming less than the TDI value (6/31, 19.4%) and the group consuming equal to or greater than the TDI value (17/98, 17.3%). In addition, no abnormal change related to cinnamon bark was found for individual patients. This paper was done to assess the risk of hepatotoxicity by the coumarin contained in Kampo medicines and to clarify whether or not the Kampo preparations in general use that contain cinnamon bark may be safely used in clinical practice.

Detection of Human Parechoviruses in Clinical and Municipal Wastewater Samples in Miyagi, Japan, in 2012-2014.

In order to study the epidemiology of human parechovirus (HPeV) infections and to evaluate the feasibility of environmental surveillance, we analyzed 281 stool samples, 265 nasopharyngeal swab samples, and 79 municipal wastewater samples for HPeV. The samples were collected in Miyagi Prefecture, Japan, between April 2012 and March 2014. HPeV was detected by reverse-transcription-PCR targeting the partial 5'-untranslated region and was genotyped by sequencing the capsid VP1 region. Seven stool samples (2.5%) and 1 nasopharyngeal swab sample (0.4%), all of which were from children under 2 years old, and 14 wastewater samples (18%) were positive for HPeV. Clear seasonality was observed: all positive samples were collected between July and December during the study period. All strains detected in the stool and wastewater samples had genotype HPeV1, and the strain from the nasopharyngeal swab sample had genotype HPeV6. A phylogenetic analysis revealed that all HPeV1 strains from the stool samples cluster together with those from the wastewater samples, indicating that the HPeV1 strains circulating in human populations can also be detected in municipal wastewater.

Semisynthesis of salviandulin E analogues and their antitrypanosomal activity.

A series of analogues of salviandulin E, a rearranged neoclerodane diterpene originally isolated from Salvia leucantha (Lamiaceae), were prepared and their in vitro activity against Trypanosoma brucei brucei was evaluated with currently used therapeutic drugs as positive controls. One of the 19 compounds prepared and assayed in the present study, butanoyl 3,4-dihydrosalviandulin E analogue was found to be a possible candidate for an antitrypanosomal drug with fairly strong antitrypanosomal activity and lower cytotoxicity.

Epigenetic and transcriptional features of the novel human imprinted lncRNA GPR1AS suggest it is a functional ortholog to mouse Zdbf2linc.

Long non-coding RNAs (lncRNAs), transcribed from the intergenic regions of animal genomes, play important roles in key biological processes. In mice, Zdbf2linc was recently identified as an lncRNA isoform of the paternally expressed imprinted Zdbf2 gene. The functional role of Zdbf2linc remains undefined, but it may control parent-of-origin-specific expression of protein-coding neighbors through epigenetic modification in cis, similar to imprinted Nespas, Kcnq1ot1 and Airn lncRNAs. Here, we identified a novel imprinted long-range non-coding RNA, termed GPR1AS, in the human GPR1-ZDBF2 intergenic region. Although GPR1AS contains no human ZDBF2 exons, this lncRNA is transcribed in the antisense orientation from the GPR1 intron to a secondary, differentially methylated region upstream of the ZDBF2 gene (ZDBF2 DMR), similar to mouse Zdbf2linc. Interestingly, GPR1AS/Zdbf2linc is exclusively expressed in human/mouse placenta with paternal-allele-specific expression and maternal-allele-specific promoter methylation (GPR1/Gpr1 DMR). The paternal-allele specific methylation of the secondary ZDBF2 DMR was established in human placentas as well as somatic lineage. Meanwhile, the ZDBF2 gene showed stochastic paternal-allele-specific expression, possibly methylation-independent, in placental tissues. Overall, we demonstrated that epigenetic regulation mechanisms in the imprinted GPR1-GPR1AS-ZDBF2 region were well-conserved between human and mouse genomes without the high sequence conservation of the intergenic lncRNAs. Our findings also suggest that lncRNAs with highly conserved epigenetic and transcriptional regulation across species arose by divergent evolution from a common ancestor, if they do not have identical exon structures.