Dengue is caused by four genetically distinct viral serotypes, dengue virus (DENV) 1-4. Following transmission by Aedes mosquitoes, DENV can cause a broad spectrum of clinically apparent disease ranging from febrile illness to dengue hemorrhagic fever and dengue shock syndrome. Progress in the understanding of different dengue serotypes and their impacts on specific host-virus interactions has been hampered by the scarcity of tools that adequately reflect their antigenic and genetic diversity. To bridge this gap, we created and characterized infectious clones of DENV1-4 originating from South America, Africa, and Southeast Asia. Analysis of whole viral genome sequences of five DENV isolates from each of the four serotypes confirmed their broad genetic and antigenic diversity. Using a modified circular polymerase extension reaction (CPER), we generated de novo viruses from these isolates. The resultant clones replicated robustly in human and insect cells at levels similar to those of the parental strains. To investigate in vivo properties of these genetically diverse isolates, representative viruses from each DENV serotype were administered to NOD Rag1-/-, IL2rgnull Flk2-/- (NRGF) mice, engrafted with components of a human immune system. All DENV strains tested resulted in viremia in humanized mice and induced cellular and IgM immune responses. Collectively, we describe here a workflow for rapidly generating de novo infectious clones of DENV - and conceivably other RNA viruses. The infectious clones described here are a valuable resource for reverse genetic studies and for characterizing host responses to DENV in vitro and in vivo.
In the current study, molecular, biological, and antigenic analyses were performed to characterize Border disease virus (BDV) strain FNK2012-1 isolated from a pig in 2012 in Japan. The complete genome comprises 12,327 nucleotides (nt), including a large open reading frame of 11,685 nt. Phylogenetic analysis revealed that FNK2012-1 was clustered into BDV genotype 1 with ovine strains. FNK2012-1 grew in porcine, bovine, and ovine primary cells and cell lines, but grew better in bovine and ovine cells than in porcine cells. Specific pathogen-free pigs inoculated with FNK2012-1 did not show any clinical signs. Noninoculated contact control pigs also did not show clinical signs and did not seroconvert. The results suggest that FNK2012-1 may be of ruminant origin and is poorly adapted to pigs. Such observations can provide important insights into evidence for infection and transmission of BDV, which may be of ruminant origin, among pigs.
SUMMARY The majority of SARS-CoV-2 infections among healthy individuals result in asymptomatic to mild disease. However, the immunological mechanisms defining effective lung tissue protection from SARS-CoV-2 infection remain elusive. Unlike mice solely engrafted with human fetal lung xenograft (fLX), mice co-engrafted with fLX and a myeloid-enhanced human immune system (HNFL mice) are protected against SARS-CoV-2 infection, severe inflammation, and histopathology. Effective control of viral infection in HNFL mice associated with significant macrophage infiltration, and the induction of a potent macrophage-mediated interferon response. The pronounced upregulation of the USP18-ISG15 axis (a negative regulator of IFN responses), by macrophages was unique to HNFL mice and represented a prominent correlate of reduced inflammation and histopathology. Altogether, our work shed light on unique cellular and molecular correlates of lung tissue protection during SARS-CoV-2 infection, and underscores macrophage IFN responses as prime targets for developing immunotherapies against coronavirus respiratory diseases. HIGHLIGHTS Mice engrafted with human fetal lung xenografts (fLX-mice) are highly susceptible to SARS-CoV-2. Co-engraftment with a human myeloid-enriched immune system protected fLX-mice against infection. Tissue protection was defined by a potent and well-balanced antiviral response mediated by infiltrating macrophages. Protective IFN response was dominated by the upregulation of the USP18-ISG15 axis.
Background: The incidence of non-B non-C hepatocellular carcinoma (NBNC-HCC), which is negative for hepatitis B surface antigen and hepatitis C virus antibodies, is on the rise. Relatively high numbers of NBNC-HCC patients are hepatitis B core antibody (HBcAb) positive, suggesting that previous HBV infection may play a role in NBNC-HCC development, though the exact mechanisms are unclear. This study aimed to investigate whether HBV genomes are integrated into the host genome of HBcAb-positive NBNC-HCC cases and how these integrations may contribute to cancer development and progression. Methods: HBV detection PCR using HBV-specific primers on DNA extracted from HBcAb-positive NBNC-HCC tissue samples was performed. Positive samples were further examined for HBV integration sites using viral DNA-capture sequencing. Additionally, hepatitis B core-related antigen (HBcrAg) serum levels were measured to assess whether they could be predictive for HBV detection PCR results. Results: Among 90 HBcAb-positive NBNC-HCC samples, HBV genome amplification was detected in 18 samples, and elevated HBcrAg levels were associated with the HBV detection PCR results. Seventeen of these samples exhibited HBV integration. The HBV genome was integrated near the TERT gene in 7 samples, resulting in significantly increased TERT mRNA levels; in the KMT2B gene (2 samples); and downstream of LOC441666 (2 samples). Conclusion: The integration sites we identified in our samples have been previously reported in HBV-related HCC, suggesting that HBV integration may also contribute to hepatocarcinogenesis in HBcAb-positive NBNC-HCC. Furthermore, HBcrAg could serve as a potential, noninvasive marker for detecting HBV integration in these cases.
As of November, 2023, SARS-CoV-2 XBB variants, including EG.5.1 (XBB.1.9.2.5.1), the currently predominant lineage, have been circulating worldwide, according to Nextstrain datasets. The EG.5.1 strain has a characteristic amino acid substitution in the spike protein (S; S:F456L), which allows the strain to escape humoral immunity (appendix p 16).1Kaku Y Kosugi Y Uriu K et al.Antiviral efficacy of the SARS-CoV-2 XBB breakthrough infection sera against omicron subvariants including EG.5.Lancet Infect Dis. 2023; 23: e395-e396Summary Full Text Full Text PDF PubMed Google Scholar EG.5.1 has further evolved, and its descendant lineage harbouring the S:L455F (ie, EG.5.1+S:L455F) variant has emerged and has been named HK.3 (XBB.1.9.2.5.1.1.3). HK.3 was initially discovered in east Asia and is rapidly spreading worldwide. Notably, the XBB subvariants bearing both S:L455F and S:F456L substitutions, including HK.3, are defined as FLip variants. These FLip variants, including JG.3 (XBB.1.9.2.5.1.3.3), JF.1 (XBB.1.16.6.1), and GK.3 (XBB.1.5.70.3) have emerged concurrently, suggesting that the acquisition of these two substitutions confers a growth advantage to XBB in the human population.2Ito J Suzuki R Uriu K et al.Convergent evolution of SARS-CoV-2 omicron subvariants leading to the emergence of BQ.1.1 variant.Nat Commun. 2023; 14: 2671Crossref PubMed Scopus (19) Google Scholar,3Bloom JD Neher RA Fitness effects of mutations to SARS-CoV-2 proteins.Virus Evol. 2023; 9vead055Crossref Scopus (2) Google Scholar We investigated the virological properties of HK.3 as a representative of the FLip variants. We estimated the relative effective reproduction number (Re) of HK.3 on the basis of genome surveillance data obtained from 13 countries reporting the substantial presence of HK.3 with a Bayesian hierarchical multinomial logistic regression model (appendix pp 9–14, 16).4Yamasoba D Kimura I Nasser H et al.Virological characteristics of the SARS-CoV-2 omicron BA.2 spike.Cell. 2022; 185: 2103-2115.e19Summary Full Text Full Text PDF PubMed Scopus (149) Google Scholar The global mean Re for HK.3 was 1·29 times higher than that of XBB.1.5 and 1·12 higher than that of EG.5.1, suggesting that HK.3 might soon become the predominant lineage worldwide. As of Oct 15, 2023, the HK.3 variant has outcompeted EG.5.1 in countries such as Australia, China, South Korea, and Singapore (appendix p 16). Next, to identify whether the enhanced infectivity of HK.3 contributes to its higher Re, we constructed lentivirus-based pseudoviruses carrying the S proteins XBB.1.5, EG.5.1, HK.3, and an XBB.1.5 derivative, XBB.1.5+L455F. Although the S:L455F substitution significantly increased the infectivity of XBB.1.5, the infectivity of HK.3 (identical to EG.5.1+S:L455F) was similar to that of EG.5.1 (appendix p 16). The difference in the effect of S:L455F between XBB.1.5 and EG.5.1 might be attributed to the epistatic effects due to the S protein structures of XBB.1.5 and EG.5.1. These results suggest that the increased Re of HK.3 is not owing to the increased infectivity caused by S:L455F. We then performed a neutralisation assay using breakthrough infection serum samples (XBB.1.5 [n=20], XBB.1.9 [n=15], XBB.1.16 [n=20], or EG.5.1 [n=18]) to address whether HK.3 evades the antiviral response of humoral immunity induced by breakthrough infection of these variants. The 50% neutralisation titre (NT50) for all breakthrough infection serum samples tested against XBB.1.5+S:L455F was significantly lower than that observed against the parental XBB.1.5 strain (appendix p 16). Notably, the NT50 for EG.5.1 breakthrough infection serum samples against HK.3 was significantly lower (1·6 times, p=0·0003) than that observed against EG.5.1 (appendix p 16). Thus, the increased Re of HK.3 might be partly attributed to the enhanced immune evasion from humoral immunity elicited by breakthrough infection subvariants of XBB, including EG.5.1, its ancestor. S:L455F is a key mutation leading to this immune evasion. JI has received consulting fees and honoraria for lectures from Takeda Pharmaceutical. KSat has received consulting fees from Moderna Japan and Takeda Pharmaceutical and has received honoraria for lectures from Gilead Sciences, Moderna Japan, and Shionogi & Co. All other authors declare no competing interests. YKo, AP, and OP contributed equally. This work was supported in part by the Japan Agency for Medical Research and Development (AMED) Strategic Center of Biomedical Advanced Vaccine Research and Development for Preparedness and Response (SCARDA) Japan Initiative for World-leading Vaccine Research and Development Centers UTOPIA (JP223fa627001, to KSat), AMED SCARDA Programme on R&D of New Generation Vaccine including New Modality Application (JP223fa727002, to KSat); AMED Research Programme on Emerging and Re-emerging Infectious Diseases (JP22fk0108146, to KSat; JP21fk0108494, to G2P-Japan Consortium and KSat; JP21fk0108425, to KSat; JP21fk0108432, to KSat; JP22fk0108511, to G2P-Japan Consortium and KSat; JP22fk0108516, to KSat; JP22fk0108506, to KSat); AMED Research Programme on HIV/AIDS (JP22fk0410039, to KSat); JST PRESTO (JPMJPR22R1, to JI); JST CREST (JPMJCR20H4, to KSat); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (23K14526, to JI); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to KSat); JSPS Research Fellow DC2 (22J11578, to KU); JSPS Research Fellow DC1 (23KJ0710, to YKo); The Tokyo Biochemical Research Foundation (to KSat); and The Mitsubishi Foundation (to KSat). Members of the G2P-Japan Consortium are listed in the appendix (p 18). Download .pdf (.63 MB) Help with pdf files Supplementary appendix
Hepatitis C virus (HCV) infection is known to induce autophagy, but the mechanism of autophagy induced by HCV remains controversial. Here, we investigated the characteristics of autophagy induced by HCV infection. First, to examine the involvement of autophagy-related gene (ATG) proteins in HCV-induced LC3 lipidation, we established ATG5, ATG13 or ATG14 knockout (KO) Huh7.5.1 cell lines and confirmed that the accumulation of lipidated LC3 was induced in an ATG13- and ATG14-independent manner. On the other hand, HCV infectivity was not influenced by deficiencies in these genes. We also confirmed that LC3-positive dots were co-localized with ubiquitinated aggregates, and deficiency of ATG5 or ATG14 enhanced the accumulation of ubiquitinated aggregates compared to that in the restored cells, suggesting that HCV infection induces ATG5- and ATG14-dependent selective autophagy. Moreover, LC3-positive ubiquitinated aggregates accumulated near the site of the replication complex. We further examined autophagy flux in cells replicating HCV RNA using bafilomycin or E64d, and found that the increase of LC3 lipidation by treatment with bafilomycin or E64d was impaired in HCV-replicating cells, suggesting that autophagy flux is inhibited by the progress of HCV infection. Our present study suggests that (1) HCV RNA replication induces selective autophagy and (2) the progress of HCV infection impairs autophagy flux.
To investigate the biocontrol capability of the entomopathogenic fungus Purpureocillium takamizusanense, the genome of the wild-type strain isolated from synnemata on Meimuna opalifera, was sequenced using a combination of HiSeq and Nanopore technologies, and annotated using evidence from RNA sequences and protein sequences from its sister species Purpureocillium lilacinum.
Since 2019, SARS-CoV-2 has undergone mutations, resulting in pandemic and epidemic waves. The SARS-CoV-2 spike protein, crucial for cellular entry, binds to the ACE2 receptor exclusively when its receptor-binding domain (RBD) adopts the up-conformation. However, whether ACE2 also interacts with the RBD in the down-conformation to facilitate the conformational shift to RBD-up remains unclear. Herein, we present the structures of the BA.2.86 and the JN.1 spike proteins bound to ACE2. Notably, we successfully observed the ACE2-bound down-RBD, indicating an intermediate structure before the RBD-up conformation. The wider and mobile angle of RBDs in the up-state provides space for ACE2 to interact with the down-RBD, facilitating the transition to the RBD-up state. The K356T, but not N354-linked glycan, contributes to both of infectivity and neutralizing-antibody evasion in BA.2.86. These structural insights the spike-protein dynamics would help understand the mechanisms underlying SARS-CoV-2 infection and its neutralization.
In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
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