記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Microbiology and Immunology  

In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population.

DOI： 10.1111/1348-0421.13165

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Although the mRNA SARS-CoV-2 vaccine has improved the mortality rate in the general population, its efficacy against rapidly mutating virus strains, especially in kidney transplant recipients, remains unclear. We examined the anti-SARS-CoV-2 spike protein IgG antibody and neutralizing antibody titers and cellular immunity against B.1.1, BA.1, and BA.5 antigens in 73 uninfected kidney recipients and 16 uninfected healthy controls who received three doses of an mRNA SARS-CoV-2 vaccine. The IgG antibody titers were significantly lower in recipients than in healthy controls. Similarly, neutralizing antibody titers against three viral variants were significantly lower in recipients. When the virus was mutated, the neutralizing antibody titers decreased significantly in both groups. In cellular immunity analysis, the number of spike-specific CD8 + non-naïve T cells against three variants significantly decreased in recipients. Conversely, the frequency of spike-specific Th2 CD4 + T-cells in recipients was higher than that in healthy controls. Nineteen recipients and six healthy controls also received a bivalent omicron-containing booster vaccine, leading to increase IgG and neutralizing antibody titers in both groups. After that, eleven recipients and five healthy controls received XBB.1.5 monovalent vaccines, increasing the neutralizing antibody titers against not only XBB.1.5, but also EG.5.1 and BA.2.86 antigens in kidney recipients. Although kidney recipients did not gain sufficient immunity against Omicron BA.5 with the third dose of vaccine, humoral response against mutant SARS-CoV-2 lineages significantly increased after bivalent Omicron-containing booster vaccine and the XBB.1.5 monovalent vaccine. Therefore, it is important for kidney recipients to continue to administer updated vaccines.

DOI： 10.1038/s41598-024-63147-z

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その他リンク： https://www.researchsquare.com/article/rs-3857039/v1.html

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Iscience  

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

DOI： 10.1016/j.isci.2024.109647

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Virology  

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

DOI： 10.1128/jvi.01638-23

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Host and Microbe  

In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both in vitro and in vivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.

DOI： 10.1016/j.chom.2024.01.001

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology  

Some heterotrophic microorganisms carry out nitrification to produce nitrite and nitrate from pyruvic oxime. Pyruvic oxime dioxygenase (POD) is an enzyme that catalyzes the degradation of pyruvic oxime to pyruvate and nitrite from the heterotrophic nitrifying bacterium Alcaligenes faecalis. Sequence similarity searches revealed the presence of genes encoding proteins homologous to A. faecalis POD in bacteria of the phyla Proteobacteria and Actinobacteria and in fungi of the phylum Ascomycota, and their gene products were confirmed to have POD activity in recombinant experiments. Phylogenetic analysis further classified these POD homologs into three groups. Group 1 POD is mainly found in heterotrophic nitrifying Betaproteobacteria and fungi, and is assumed to be involved in heterotrophic nitrification. It is not clear whether group 2 POD, found mainly in species of the Gammaproteobacteria and Actinobacteria, and group 3 POD, found simultaneously with group 1 POD, are involved in heterotrophic nitrification. The genes of bacterial group 1 POD comprised a single transcription unit with the genes related to the metabolism of aromatic compounds, and many of the genes group 2 POD consisted of a single transcription unit with the gene encoding the protein homologous to 4-hydroxy-tetrahydrodipicolinate synthase (DapA). LysR- or Cro/CI-type regulatory genes were present adjacent to or in the vicinity of these POD gene clusters. POD may be involved not only in nitrification, but also in certain metabolic processes whose functions are currently unknown, in coordination with members of gene clusters.

DOI： 10.1007/s10482-023-01862-9

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その他リンク： https://link.springer.com/article/10.1007/s10482-023-01862-9/fulltext.html

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-021-02579-3

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep39991

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