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The P gene of paramyxoviruses is unique in producing not only P but also "accessory" C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C', C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported. [ABSTRACT FROM AUTHOR]

Abstract: The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release. [ABSTRACT FROM AUTHOR]

Abstract: The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-β induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-α/β receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN. [Copyright &y& Elsevier]

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted. [Copyright &y& Elsevier]

Investigates the active subunit stoichiometry of the influenza virus A ion channel M2 by expressing mixed oligomers of M2 and adamantine-resistant M2 subunits. Protein expression in mammalian cells and oocytes; Subunit composition of ion channel; Membrane current measurements.

Reports that high level expression of the M2 ion channel protein of influenza virus inhibits the rate of intracellular transport of the influenza virus hemagglutinin (HA) and that of other integral membrane glycoproteins. Flow cytometry; Proteinase digestion of neutral pH and low pH forms of HA; Glycosidase digestions; Chemical cross-linking.

Abstract: Optineurin has been shown to be involved in primary open-angle glaucoma. We recently found that optineurin is involved in familial amyotrophic lateral sclerosis (ALS). On the other hand, optineurin has been shown to inhibit transcription factors related to innate immunity such as NF-κB and interferon regulatory factor-3 (IRF3). In the present study, the effect of ALS-associated optineurin mutations on IRF3 activation was investigated. Optineurin inhibited IRF3 activation induced by melanoma differentiation-associated gene 5 or Toll-IL-1 receptor domain-containing adaptor-inducing interferon-β. The inhibition was abrogated by mutations related to ALS but not by a mutation related to glaucoma. Reporter assay indicated that the JAK-STAT signaling pathway was not affected by optineurin. These results show that ALS-related optineurin is involved in the IRF3 activation pathway. Pathogenesis of ALS may be associated with some kind of innate immunity, especially that against virus infection, through IRF3 activation. [Copyright &y& Elsevier]

Umifenovir is a broad‐spectrum antiviral agent used to treat influenza in China and Russia, and it has been studied as an antiviral agent for the treatment of coronavirus disease 2019 (COVID‐19). We have previously reported the synthesis of novel umifenovir analogues and their biological evaluation with a focus on their inhibitory activity against the binding of the spike glycoprotein (S‐protein) of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and the angiotensin‐converting enzyme 2 (ACE2) receptor; however, no strong inhibitory activity was observed from these analogues. In the present study, an additional set of umifenovir analogues was synthesized with replacement of the substituents at the 2‐, 3‐, and 4‐positions of the indole, and a cell‐based assay using SARS‐CoV‐2 (B.1.1) was performed to examine the antiviral activity of the analogues. We found that one of the newly synthesized umifenovir analogues exhibited antiviral activity and reduced the viral load to 0.06 % as compared to the control when it was assessed in the presence of nafamostat and marimastat, which inhibit cell‐surface viral entry. In contrast, when this analogue was evaluated without the addition of nafamostat or marimastat, it exhibited less antiviral activity, suggesting that the umifenovir analogue would exert antiviral activity mainly by inhibiting endosome‐mediated viral entry. [ABSTRACT FROM AUTHOR]

Chlorous acid water (HClO2) is known for its antimicrobial activity. In this study, we attempted to accurately assess the ability of chlorous acid water to inactivate SARS-CoV-2. When using cell culture supernatants of infected cells as the test virus, the 99% inactivation concentration (IC99) for the SARS-CoV-2 D614G variant, as well as the Delta and Omicron variants, was approximately 10ppm of free chlorine concentration with a reaction time of 10 minutes. On the other hand, in experiments using a more purified virus, the IC99 of chlorous acid water was 0.41–0.74ppm with a reaction time of 1 minute, showing a strong inactivation capacity over 200 times. With sodium hypochlorite water, the IC99 was 0.54ppm, confirming that these chlorine compounds have a potent inactivation effect against SARS-CoV-2. However, it became clear that when using cell culture supernatants of infected cells as the test virus, the effect is masked by impurities such as amino acids contained therein. Also, when proteins (0.5% polypeptone, or 0.3% BSA + 0.3% sheep red blood cells, or 5% FBS) were added to the purified virus, the IC99 values became high, ranging from 5.3 to 76ppm with a reaction time of 10 minutes, significantly reducing the effect. However, considering that the usual usage concentration is 200ppm, it was shown that chlorous acid water can still exert sufficient disinfection effects even in the presence of proteins. Further research is needed to confirm the practical applications and effects of chlorous acid water, but it has the potential to be an important tool for preventing the spread of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) and the auxiliary receptor Neuropilin-1 (NRP1) to enter host cells. NRP1 has another isoform, NRP2, whose function in COVID-19 has seldom been reported. In addition, although patients with severe cases of COVID-19 often exhibit increased levels of proinflammatory cytokines, the relationship between these cytokines and SARS-CoV-2 proliferation remains unknown. The aim of this study is to clarify the roles of proinflammatory cytokines in Neuropilin expressions and in SARS-CoV-2 infection. To identify the expression patterns of NRP under inflamed and noninflamed conditions, next-generation sequencing (RNA-seq), immunohistochemistry, quantitative real-time PCR, and Western blotting were performed using primary cultured fibroblast-like synoviocytes, MH7A (immortalized cell line of human rheumatoid fibroblast-like synoviocytes), immortalized MRC5 (human embryonic lung fibroblast), and synovial tissues. To measure viral proliferative capacity, SARS-CoV-2 infection experiments were also performed. NRP2 was upregulated in inflamed tissues. Cytokine-stimulated human fibroblast cell lines, such as MH7A and immortalized MRC5, revealed that NRP2 expression increased with co-stimulation of tumor necrosis factor α (TNFα) and interleukin-1 beta (IL-1β) and was suppressed with anti-TNFα antibody alone. TNFα and IL-1β promoted SARS-CoV-2 proliferation and Spike protein binding. The viral proliferation coincided with the expression of NRP2, which was modulated through plasmid transfections. Our results revealed that proinflammatory cytokines, including TNFα, contribute to NRP2 upregulation and SARS-CoV-2 proliferation in host human cells. [ABSTRACT FROM AUTHOR]

Background: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNβ), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNβ than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNβ overproduction during optineurin deficiency both in vitro and in vivo. Methods: To investigate the mechanism of IFNβ overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. Results: IFNβ overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNβ overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNβ overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. Conclusion: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNβ overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases. [ABSTRACT FROM AUTHOR]

The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix. [ABSTRACT FROM AUTHOR]

The decrease of antibody efficacy to mutated SARS-CoV-2 spike RBD explains the breakthrough infections and reinfections by Omicron variants. Here, we analyzed broadly neutralizing antibodies isolated from long-term hospitalized convalescent patients of early SARS-CoV-2 strains. One of the antibodies named NCV2SG48 is highly potent to broad SARS-CoV-2 variants including Omicron BA.1, BA.2, and BA.4/5. To reveal the mode of action, we determined the sequence and crystal structure of the Fab fragment of NCV2SG48 in a complex with spike RBD from the original, Delta, and Omicron BA.1. NCV2SG48 is from a minor VH but the multiple somatic hypermutations contribute to a markedly extended binding interface and hydrogen bonds to interact with conserved residues at the core receptor-binding motif of RBD, which efficiently neutralizes a broad spectrum of variants. Thus, eliciting the RBD-specific B cells to the longitudinal germinal center reaction confers potent immunity to broad SARS-CoV-2 variants emerging one after another. Broadly neutralizing antibodies to SARS-CoV-2 variants including Omicron were isolated from long-term hospitalized convalescent patients with early SARS-CoV-2 strain B.1.1, and the mechanisms are identified from crystal structures. [ABSTRACT FROM AUTHOR]

• Virus from swab sample S66 in hiroshima had T-base insertions in the ORF8 region. • Cloning, and TIDE analysis confirmed mixed viruses with different T-base insertions. • Virus with one t (T1+) was 70–75 %; others included T0, T2+, T3+, T4+, and T5+. • C27911T point mutation led to a sequence of seven or more consecutive t bases. • Study highlights a potential mechanism for insertion mutations in SARS-CoV-2. The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains. [ABSTRACT FROM AUTHOR]

• Viable SARS-CoV-2 can be detected in the air around patients with an early infection. • Airborne viable SARS-CoV-2 is associated with high levels of airborne viral RNA. • Airborne SARS-CoV-2 is not related to nasopharyngeal viral RNA and viable virus. This study assessed the concentration of SARS-CoV-2 in the air of hospital rooms occupied by patients with COVID-19 who had viable SARS-CoV-2 in nasopharyngeal (NP) samples in early infection. Between July and October 2021, NP swabs were collected from 20 patients with early SARS-CoV-2 infection admitted to a tertiary hospital in Japan. Air samples were collected from their rooms, tested for SARS-CoV-2 RNA, and cultured to determine potential infectivity. The NP swab samples of 18 patients were positive for viable SARS-CoV-2 (median concentration: 4.0 × 105 tissue culture infectious dose 50/ml). In the air samples, viral RNA (median concentration: 1.1 × 105 copies/m3) was detected in 12/18 (67%) patients, and viable virus (median concentration: 8.9 × 102 tissue culture infectious dose 50/m3) was detected in 5/18 (28%) patients. The median time between illness onset and sampling was 3 days. The RNA concentration was significantly higher in samples wherein viable SARS-CoV-2 was detected than in samples in which viable virus was not detected (P -value = 0.027). Viable SARS-CoV-2 can be detected in the air surrounding patients with early SARS-CoV-2 infection. Health care workers should pay attention to infection control when caring for patients with early SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

TONS504 (C51H58N8O5I2), a chlorine derivative, effectively generates singlet oxygen by light activation and exhibits photodynamic antimicrobial effects (PAEs) on various pathogens. However, this photosensitizer has some limitations: a high tendency to self-aggregate and a relatively weak PAE for Gram-negative bacteria compared with Gram-positive bacteria. To overcome these limitations, the present study investigated the synergistic effects of the PAE of TONS504 and two additives commonly contained in ophthalmic solutions: benzalkonium chloride (BAC) or ethylenediaminetetraacetic acid (EDTA). Staphylococcus aureus and Pseudomonas aeruginosa were exposed to TONS504 and/or each additive. Photodynamic antimicrobial chemotherapy was performed with light irradiation centered at a wavelength of 665 nm with a total light energy of 30 J/cm2. Following incubation, the number of colonies formed was counted. Additionally, we examined the inhibitory effects of the additives on TONS504 self-aggregation by observing its absorption spectrum. Consequently, the PAEs of TONS504 on S. aureus were enhanced by both additives, and BAC displayed stronger synergistic effects on the bacteria than EDTA. By contrast, only EDTA increased the PAE on P. aeruginosa. The peak of the TONS504 absorption spectrum shifted to a longer wave length and the absorbance increased in the presence of BAC, suggesting that BAC inhibited the self-aggregation of the photosensitizer. In conclusion, the combination of BAC or EDTA and TONS504-mediated photodynamic antimicrobial chemotherapy exhibits a synergistic antimicrobial effect on S. aureus and P. aeruginosa. The optimal additive to enhance the PAE may differ between bacterial strains. [ABSTRACT FROM AUTHOR]

The solid‐state thermal structure transformation of methylammonium vanadate, (CH3NH3)VO3, from −150 °C to 350 °C is reported. Variable‐temperature X‐ray single‐crystal structure analysis at 23, 0, −50, −100, and −150 °C reveal (CH3NH3)VO3 comprises of methylammonium cations and "snake‐like" ([VO3]−)n anion chains propagating along the c‐direction in the Pna21 space group. In between −150 and −100 °C, we observe a reversible structural transformation due to the re‐orientation of the methylammonium cations in the crystal packing, which is also confirmed by the reversible profiles observed in differential scanning calorimetry. The methylammonium vanadate is stable until at ca. 100 °C and further heating releases methylamine and water and V2O5 is formed at ca. 275 °C. Furthermore, we show that the methylammonium vanadate can be used as a negative staining reagent for visualizing SARS‐CoV‐2, allowing us to discern the spike proteins from the body of the virus using transmission electron microscopy. [ABSTRACT FROM AUTHOR]

New treatment methods, such as REGN-CoV2, have been approved for patients with coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the effect of the drug on the duration of infectious viral shedding and viral mutations is unknown. In this study, we investigated the clinical efficacy of REGN-CoV2 treatment in patients with mild to moderate disease and compared its antiviral effects against different strains of SARS-CoV-2. Viral culture and PCR testing were performed on the pharyngeal swabs collected from 28 patients with COVID-19 who were admitted and treated at Hiroshima University Hospital during the study period. Of these, 23 patients were treated with REGN-CoV2. The patients were classified into the REGN-CoV2(+) and REGN-CoV2(−) groups, and the clinical course was compared between the groups. The 50% inhibitory concentrations (IC 50) of REGN-CoV2 against the isolated virus strains were determined. After treatment with REGN-CoV2, the virus culture positivity rate was greatly reduced. The time to negative viral culture was significantly shorter in the REGN-CoV2(+) group than in the REGN-CoV2(−) group. In vitro evaluation of REGN-CoV2 against isolated virus strains also showed efficacy. REGN-CoV2 treatment was effective in patients with mild COVID-19 and could shorten the period of infectious viral shedding. This may be an important factor in preventing the spread of infection. It may be possible to revise the isolation period for patients with mild disease treated with REGN-CoV2. [ABSTRACT FROM AUTHOR]

Abstract: Sendai virus (SeV) M protein has a YLDL motif, which is essential for budding of virus-like particles (VLPs) by expression of the M protein. We investigated the importance of the YLDL motif for SeV budding. Virus budding of an M-deficient SeV was not rescued by transient expression of motif mutants, M-A2 (ALDA) and M-A4 (AAAA), and viruses possessing those mutations hardly propagated in cultured cells. However, a budding-competent revertant virus, SeV M-A2R, was obtained from SeV M-A2, and nucleotide sequencing showed an ALDV sequence at the motif instead of the ALDA sequence derived from M-A2. The M-A2R protein rescued budding of an M-deficient SeV, formed VLPs when expressed with viral C protein, and restored the capacity to bind with Alix/AIP1. The results indicate that the YLDL motif is essential for efficient budding in the context of virus infection and suggest involvement of Alix/AIP1 in SeV budding. [ABSTRACT FROM AUTHOR]

This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic. [ABSTRACT FROM AUTHOR]

Approximately 5% of patients with coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 develop severe COVID-19. Severe COVID-19 requires respiratory management with mechanical ventilation and an extended period of treatment. Prolonged infectious virus shedding is a concern in severe COVID-19 cases, but few reports have examined the duration of infectious virus shedding. Therefore, we investigated the duration of infectious virus shedding in patients transferred to Hiroshima University Hospital with severe COVID-19 requiring mechanical ventilation. Nasopharyngeal swab specimens were collected and analyzed using both viral culture and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) tests between December 2020 and February 2021. Of the 23 patients tested, the proportions of those with positive test results at first specimen collection (the median number of days to first specimen collection after symptom onset was 10) on RT-qPCR and viral culture tests were 95·7% and 30·4%, respectively. All six patients with positive viral culture test results who were followed-up tested negative 24 days after symptom onset but remained positive on RT-qPCR. Viral loads based on PCR testing did not decrease over time, but those determined via culture tests decreased over time. The longest negative conversion time was observed in a dialysis patient on immunosuppressive drugs. This study indicated that patients with severe COVID-19 remain culture positive for ≥ 10 days after symptom onset. Additionally, immunosuppressed patients with severe COVID-19 could consider isolation for ≥ 20 days. [ABSTRACT FROM AUTHOR]

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 μM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)—a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1—similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19. [ABSTRACT FROM AUTHOR]

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells. [ABSTRACT FROM AUTHOR]

Background: In this study, we performed molecular characterization of SARS-CoV-2 strains in Hiroshima and its mutation pattern between the first and second waves of the outbreak. Method: A total of 55 nasal swab samples from the first wave in Hiroshima and 13 from the second wave were examined quantitatively by RT-qPCR and qualitatively by nested PCR using specific primers. Four samples from each wave underwent next-generation sequencing and phylogenetic tree analysis including controls and all sequences retrieved in Japan from GISAID and GenBank. Subsequently, mutations were examined. Results: Viral load ranged 7.85 × 101−1.42 × 108 copies/ml. Of 68 samples, one was Asian type-O, 65 were European type-GR, and 2 were undetectable. Phylogenetic tree analysis indicated that Japan was infected with various Asian strains (L, S, V, O) from January through April. By second week of March, European strains (G, GH, GR) had appeared, and GR strains became predominant after mid-March. The first case in Hiroshima was classified as Asian strain O, and the rest were GR strains. Then, second wave of GR strains appeared independently with 11–15 base mutations. Comparing the first- and second-wave GR strains, mutation rate was 1.17–1.36 × 10−3 base substitutions per site per year; in addition, amino acid changes occurred at S1361P and P3371S in ORF1a, A314V in ORF1b, and P151L in N. All seven GR strains were D614G variants with R202K and G203R mutations in N. A single-nucleotide insertion in ORF8 that causes a defect in ORF8 protein was found in one isolate (S66) from the second wave. Conclusion: Our findings reveal the evolutionary hierarchy of SARS-CoV-2 in Japan. The predominant D614G variants and a new form of ORF8 deletion in Hiroshima provide the clue for role of viral factor in local outbreaks of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The pathogenic filamentous fungi Fusarium solani (F. solani) and Aspergillus fumigatus (A. fumigatus) are common causes of fungal keratitis. We have here evaluated the antifungal efficacy of photodynamic antimicrobial chemotherapy (PACT) with the novel chlorin derivative TONS 504 and a light-emitting diode (LED) with a wavelength of 660 nm for these fungal species. Isolated fungal spores were irradiated at LED energies of 10, 20, or 30 J/cm2 in the presence of TONS 504 at concentrations of 1 or 10 mg/L. As a control, spores were exposed to TONS 504 or LED radiation alone. The treated spores were then cultured on potato dextrose agar plates at 25 °C for 3 to 4 days before determination of colony formation as a measure of viability. Fungal growth was inhibited in a manner dependent on both LED energy and TONS 504 concentration. The inhibitory effect on F. solani was complete with TONS 504 at a concentration of 1 mg/L and LED irradiation at 30 J/cm2 as well as at a TONS 504 concentration of 10 mg/L and LED irradiation at 10, 20, or 30 J/cm2. In contrast, that on A. fumigatus was only partial at a TONS 504 concentration of 10 mg/L and LED irradiation at 20 or 30 J/cm2. The antifungal effect of PACT on A. fumigatus was thus inferior to that on F. solani. PACT with TONS 504 and an LED thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious fungal keratitis. [ABSTRACT FROM AUTHOR]

Attempts have been made to visualize tumor cells intraoperatively with fluorescence guidance. However, the clear demarcation and complete tumor resection have always been a challenging task. To address this, we have developed a novel fluorescence bioimaging system with vesicular stomatitis virus (VSV) incorporating Katushka, near‐infrared fluorescent protein. VSV is tumor‐specific owing to the deficiency of antiviral interferon signaling pathways in tumor cells. We aimed to evaluate the tumor specificity of the recombinant VSV‐Katushka (rVSV‐K) in osteosarcoma cells and to assess the feasibility of complete tumor resection by the rVSV‐K fluorescence guidance. In in vitro experiments, mouse and human osteosarcoma cell lines and normal human mesenchymal stem cells were infected with rVSV‐K and observed by fluorescence microscopy. Near‐infrared fluorescence was observed only in osteosarcoma cells, even at a low‐concentration of virus infections. In in vivo experiments, mouse osteosarcoma (LM8) cells were transplanted subcutaneously into the back of immune‐competent mice to produce an osteosarcoma, which was then injected with rVSV‐K. The areas emitting fluorescence were resected using a bioimaging system. The distance between the surgical and tumor margins of the fluorescence‐guided resection with rVSV‐K group was significantly larger than that of the non‐guided resection groups. The local recurrence rate was significantly lower in the fluorescence‐guided resection with rVSV‐K group than in the non‐guided resection groups. The distant metastasis rate and average survival rate were not significantly different between all groups. These results suggest that the rVSV‐K is specific to osteosarcoma cells and enables complete tumor resection of osteosarcomas in mice. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [ABSTRACT FROM AUTHOR]

The terrestrial stems of Ephedra (Ephedra spp.; including Ephedra sinica Stapf and Ephedra przewalskii Stapf) extracts are used in traditional medicines in East Asia. In Japan, the Kampo formula containing E. sinica extract is prescribed for the treatment of the common cold, influenza virus infections, and mild symptoms of coronavirus disease 2019 (COVID-19). Although ephedrine alkaloids in E. sinica exert antitussive effects, they may have side effects associated with the sympathetic nervous system. E. przewalskii extract, a drug used in traditional Uyghur and Mongolian medicine, is considered to be free of ephedrine alkaloids and is a promising candidate for the treatment of infectious diseases. However, its use is currently limited because evidence of its antiviral efficacy remains inconclusive. Aim of the study : We compared the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) effects of E. przewalskii and E. sinica extracts in vitro. Additionally, we examined the differences in their antiviral effects against different SARS-CoV-2 strains. VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 (Conventional, Delta, and Omicron strains—BA.1, BA.2, BA.4, and BA.5), and lysates prepared from each herbal extract were added. The infectious titer was determined using the 50% tissue culture infectious dose (TCID 50) method; in turn, the half-maximal inhibitory concentration (IC 50) was calculated for each extract to compare the antiviral efficacy of E. sinica and E. przewalskii extracts. Further, the extracts were compared with remdesivir for their antiviral efficacy against the conventional viral strain. To verify the effect of the inactivation of virus particles, these extracts were added to each SARS-CoV-2 strain, and the infectious titers were determined using the TCID 50 method. The antiviral efficacy (i.e., IC 50) of the E. przewalskii extract against each SARS-CoV-2 strain was 2.7–10.8-fold greater than that of the E. sinica extract. The antiviral efficacy of the E. przewalskii extract against conventional viral strains was compared with that of remdesivir, which was 1/27.6 of remdesivir's efficacy. The E. sinica extract showed minimal inactivation of virus particles of each strain, whereas the E. przewalskii extract resulted in substantial viral inactivation. The E. przewalskii extract showed higher antiviral activity against SARS-CoV-2 than the E. sinica extract. Overall, our study suggests that E. przewalskii extract can be used for the treatment of viral infections, including COVID-19. [Display omitted] • E. przewalskii is free of ephedrine alkaloids and is safer for infectious diseases. • Anti-SARS-CoV-2 effects of E. przewalskii and E. sinica extracts were compared. • E. przewalskii extract shows 2.7–10.8-fold greater antiviral activity than E. sinica. • E. przewalskii extract shows promise against viral infections, including COVID-19. [ABSTRACT FROM AUTHOR]

An influenza epidemic is still a problem despite the development of vaccines and anti-influenza drugs. Preventive measures such as handwashing are fundamental and important for counteracting influenza virus infection. In this study, we clarified the anti-influenza virus effects of surfactants, which are the main components of hand soaps for hand washing: potassium oleate (C18:1), sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS). For a human influenza virus strain (H3N2), C18:1 reduced the infectivity by 4 logs or more, whereas LES and SDS reduced the infectivity by 1 log or less. Similar results were obtained when an avian influenza virus strain (H5N3) was used. The interaction between the surfactant and virus was then investigated by isothermal titration calorimetry. The LES-virus system showed a positive value of enthalpy changes (ΔH), meaning an exothermic interaction that indicated a hydrophobic interaction. In contrast, both the C18:1-virus system and the SDS-virus system showed negative values of ΔH, meaning an endothermic interaction that indicated an electrical interaction. The ΔH value of the C18:1-virus system was much higher than that of the SDS-virus system. A mixture of C18:1 and HA proteins similarly showed negative values of ΔH. These results indicate that influenza virus inactivation by a hydrophobic interaction of a surfactant with the viral envelope is insufficient to prevent infection, whereas inactivation by an electrical interaction of a surfactant with HA proteins is sufficient to prevent influenza virus infection. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (P. aeruginosa) is a major cause of infectious keratitis, which itself is a major cause of blindness worldwide. We have now evaluated the time-dependent effectiveness of photodynamic antimicrobial chemotherapy (PACT) with the chlorin derivative TONS 504 and a light-emitting diode (LED) on P. aeruginosa in vitro. PACT with TONS 504 (10 mg/L) and irradiation (30 J/m2) by an LED device that delivers light centered on a wavelength of 660 nm was applied to 1 × 106 colony-forming units of P. aeruginosa in liquid medium. The bacteria were then cultured at 37 °C for various times before assay of viability by determination of colony formation on agar plates. The effect of a second irradiation at 3 h after the initial LED exposure was also examined. Bacterial growth was markedly inhibited between 3 and 9 h after PACT with TONS 504, with the maximal effect being apparent at 3 h. Furthermore, a second exposure to LED irradiation at 3 h after the first treatment enhanced the inhibitory effect on bacterial growth. PACT with TONS 504 thus inhibited the growth of P. aeruginosa in a time-dependent manner, and an additional irradiation exposure applied 3 h after the first LED treatment greatly increased the effectiveness of PACT. This antibacterial system thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious keratitis. [ABSTRACT FROM AUTHOR]

ABSTRACT: Sarcomas are associated with a high incidence of lung metastasis, which leads to a high‐risk of cancer death. This study was performed to explore the pre‐clinical theranostic potential of a novel fully functional recombinant vesicular stomatitis virus carrying imaging gene Katushka (rVSV‐K), as virotherapy and circulating tumor cells (CTCs) detection in the syngeneic mouse model of osteosarcoma with spontaneous pulmonary metastases. Recombinant VSV‐K was generated and evaluated in vitro on human and murine osteosarcoma cells. Spontaneous osteosarcoma metastases were established in immune‐competent mice by implanting subcutaneously syngeneic osteosarcoma LM8 cells. The vector was injected into the tumor‐bearing mice via jugular vein either once or repeatedly. To assess effectiveness, primary tumor growth and development of lung metastasis as well as survival were evaluated. We found that rVSV‐K efficiently replicated in and killed all osteosarcoma cell lines in time‐dependent manners. Both single or repeated systemic injections of the virus did not inhibit the growth of the primary tumor, but the repeated administration could effectively suppress the development of lung metastases and was likely responsible for the observed increase in survival. Furthermore, we demonstrated, for the first time, that CTCs in blood samples from syngeneic osteosarcoma‐bearing mice were successfully detected by utilizing rVSV‐K ex vivo. Our results show that repeated systemic injections of rVSV‐K are an effective anti‐metastatic agent against osteosarcoma in immune‐competent mice and this virus to be a useful tool for detection of osteosarcoma CTCs, suggesting that further development of future viral‐based theranostic approach in patients with osteosarcoma is warranted. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2562–2569, 2018. [ABSTRACT FROM AUTHOR]

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding. [ABSTRACT FROM AUTHOR]

Feline calicivirus (FCV) is an important veterinary pathogen that causes acute upper respiratory tract diseases and, occasionally, highly contagious febrile hemorrhagic syndrome in cats. Many viruses have adopted mechanisms for evading IFN-α/β signaling, particularly by directly or indirectly suppressing activation of IRF-3. In this study, we investigated whether nonstructural proteins of FCV possess these mechanisms. When p39, a nonstructural protein of FCV, was transiently expressed in 293T cells, it suppressed IFN-β and ISG15 mRNA production induced by dsRNA. Expression of p39 also suppressed phosphorylation and dimerization of IRF-3 induced by dsRNA. These results suggest that p39 suppresses type 1 IFN production by preventing IRF-3 activation. This may become an important factor in understanding the pathogenesis and virulence of FCV. [ABSTRACT FROM AUTHOR]

Efforts to identify improved treatments for corneal infection include the development of photodynamic antimicrobial chemotherapy (PACT). We evaluated the antimicrobial effect of PACT with a novel porphyrin derivative, TONS 504, and a novel light system on methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Bacteria were irradiated with a light-emitting diode (LED) at energies of 10, 20, or 30 J/cm in the presence of various concentrations of TONS 504. Bacterial viability was assessed at 30 min and 24 h after irradiation by determination of colony formation on agar plates. PACT inhibited the growth of both MSSA and MRSA as early as 30 min after light exposure. Complete inhibition of bacterial growth was apparent at 24 h after irradiation at a TONS 504 concentration of 1 mg/L and LED energies of ≥10 J/cm or a TONS 504 concentration of 0.5 mg/L and LED energies of ≥20 J/cm for MSSA, and at a TONS 504 concentration of 10 mg/L and LED energies of ≥10 J/cm or of a TONS 504 concentration of 1 mg/L and LED energies of ≥20 J/cm for MRSA. Bacterial growth was unaffected by TONS 504 in the absence of irradiation or by irradiation in the absence of TONS 504. Our results thus demonstrate the antimicrobial efficacy of PACT with TONS 504 and a LED against both MSSA and MRSA in vitro, and they therefore provide a basis for further investigation of this system as a potential treatment for corneal infection. [ABSTRACT FROM AUTHOR]

The Sendai virus (SeV) C proteins are shown to exert multiple functions during the course of infection. Perhaps reflecting their many functions, they occur at multiple sites of the cell. In this study, we focused on the nuclear-localizing ability of the smaller C protein, Y1, and found that this translocation is mediated by Ran GTPase but not by passive diffusion, and that basic residues within the 149-157 amino acid region are critical for that. The mechanism of inhibition of interferon (IFN)-signaling seemed to differ between the C and Y1 proteins, since deletion of 12 C-terminal amino acids resulted in a loss of the function for the C but not for the Y1 protein. The ability of Y1 mutants to inhibit IFN-α-induced, ISRE-driven expression of a reporter gene almost paralleled with that to localize in the nucleus. These results suggest that nuclear localization of the Y1 protein might be important for the inhibitory effect on type-I IFN-stimulated gene expression. [ABSTRACT FROM AUTHOR]

Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses. [ABSTRACT FROM AUTHOR]

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769−1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4 T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769−1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID. [ABSTRACT FROM AUTHOR]

A significant limitation to oncolytic virotherapy in vivo is the lack of a clinically relevant means of delivering the virus. We evaluated the oncolytic activity of vesicular stomatitis virus (VSV) in human osteosarcoma cells and explored isolated limb perfusion (ILP) as a novel oncolytic virus delivery system to extremity sarcoma in immune-competent rats. Human and rat osteosarcoma cells transduced with rVSV-lacZ uniformly expressed β-gal. VSV was fully capable of replicating its RNA genome in all osteosarcoma cell lines, and efficiently killed them in time- and dose-dependent manners, whereas normal bone marrow stromal cells were refractory to the virus. VSV delivered by ILP inhibited growth of osteosarcoma xenografts more potently than that injected intravenously and intratumorally in the hind limb of immune-competent rats. Histopathological sections of tumor lesions treated by ILP-delivered VSV showed positive for VSV-G protein. There were no VSV-G expressions in perfused leg muscle, nonperfused leg muscle, brain, lung, and liver in VSV-treated rats. Our findings show efficient VSV gene expression and replication in osteosarcoma cells, suggesting that osteosarcoma may be a promising target for oncolytic virotherapy with VSV. Furthermore, we firstly showed that ILP of VSV against extremity sarcoma caused antitumor activity. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:795-800, 2011 [ABSTRACT FROM AUTHOR]

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C′/C(-), 4C(-), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-α-mediated anti-viral state. Infection by the virus (Cm2′) containing mutations at K77 and D80 induced significant IFN-β production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2′ virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-β and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production. [ABSTRACT FROM AUTHOR]

Abstract: The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C′, C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C′ and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C′ and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection. [Copyright &y& Elsevier]

Abstract: Sendai viruses (SeV) derived from persistent infection have a capacity to interfere with co-infected wild-type virus. Here we showed that interference was also caused by the laboratory strains Z and Nagoya. The leader mutations A20U and A24U related to viral adaptation from mice to chicken eggs significantly affected the capacity for viral interference, especially through genome amplification. Furthermore, recombinant SeV that possessed the mutations A34G and G47A, which are commonly found in the leader sequence of persistent infection-derived SeV strains, had an increased capacity for interference. Viral replication of human parainfluenza viruses 1, 2, and 3, but not the mumps virus or Newcastle disease virus, was suppressed by co-infection of a persistent infection-derived SeV strain, suggesting suppression of closely related human paramyxoviruses. These results indicate that homologous interference is partly dependent on the promoter sequence and further suggest involvement of promoter activity for genome amplification related to host factors in viral interference. [Copyright &y& Elsevier]

Abstract: Sendai virus (SeV) is unique in that one of the viral accessory proteins, C, enhances budding of virus-like particles (VLPs) formed by SeV matrix protein M by physically interacting with Alix/AIP1. C protein itself does not have the ability to form VLPs, while M protein provides viral budding force, like other enveloped viruses. Here we show that SeV C protein recruits Alix/AIP1 to the plasma membrane (PM) to facilitate VLP budding. SeV M-VLP budding is sensitive to overexpression of a dominant-negative (DN) form of VPS4A only in the presence of the C proteins, which is able to recruit Alix/AIP1 to the PM. Our results indicate that SeV M and C proteins play separate roles in the budding process: M protein drives budding and C protein enhances the efficiency of the utilization of cellular MVB sorting machinery for efficient VLP budding. [Copyright &y& Elsevier]

Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 × 104 copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications. [ABSTRACT FROM AUTHOR]

Abstract: The gene encoding Sendai virus nucleocapsid protein was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. The high level of recombinant Sendai virus nucleocapsid protein expression (12–14mg/l of yeast culture) was obtained. The evaluation of recombinant proteins expression in yeast by Western blot analysis revealed specific reactivity with immune sera. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid protein. These structures contained host RNA, which was resistant to an RNase treatment. The nucleocapsid protein revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. The development of a simple, efficient and cost-effective system for generation of Sendai virus nucleocapsid protein might help to upgrade reagents for virus serology, and facilitate investigation of virus replication and RNA encapsidation mechanisms. [Copyright &y& Elsevier]

Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein–protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding. [Copyright &y& Elsevier]

Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN. [ABSTRACT FROM AUTHOR]

Metastasis to the liver remains an important problem in the treatment of patients with gastrointestinal cancer. We examined the mechanism and effect on liver metastasis of in vivo interleukin-2 (IL-2) gene transfer to the liver. RCN-9 cells derived from F344 rat colon adenocarcinoma were injected into syngeneic rats via the ileocecal vein to induce liver tumors. A total of 2.5×109 pfu of adenovirus vector harboring the human IL-2 gene (AdCMVhIL-2), or 2.5×109 pfu of control vector encoding β-galactosidase was administered before RCN-9 cell challenge. On day 14, mean tumor weight was 4.0±2.4 g in the control group, whereas IL-2–transduced livers had no tumors. Survival of AdCMVhIL-2–treated rats was significantly longer than that of control rats (P<.01). Flow cytometry demonstrated that the proportion of natural killer (NK) cells had increased among sinusoidal cells collected from IL-2–transduced livers. These cells were highly cytotoxic to RCN-9 cells in vitro in the presence of a physiological high concentration of recombinant IL-2. Preventative effects of IL-2 transduction of the liver against liver metastasis were lost after depletion of NK cells by treatment with anti–asialo GM1 antibodies. Our results indicate that IL-2 gene transfer to the liver prevents liver metastasis by continuously providing physiological high concentrations of IL-2 in the liver, thereby activating sinusoidal NK cells. Cancer Gene Therapy (2002) 9, 655–664 doi:10.1038/sj.cgt.7700483 [ABSTRACT FROM AUTHOR]

The V protein of Sendai virus (SeV) is nonessential for virus replication in cell culture but indispensable for viral pathogenicity in mice. At the C terminus of the V protein, there are amino acid residues conserved among the members of the Paramyxovinae subfamily that are clustered in three regions: region I, just downstream of the RNA editing site; and regions II and III, cysteine-rich zinc-finger-like regions. In the present study, we introduced mutations into the conserved amino acids and generated nine mutant viruses. All of the viruses had impaired virus replication in mouse lungs and attenuated virulence in mice. Furthermore, the C-terminal polypeptides fused with glutathione-S-transferase with a mutation in region I, II, or III all had impaired Zn binding in a 65Zn-binding assay in solution. These results demonstrate that the conserved amino acids are important for V protein function, probably via protein conformation dependent on Zn binding. One mutant, SeV V-H318N, had inefficient RNA editing, indicating that the nucleotide that is a part of the codon encoding histidine at position 318 is conserved for the RNA editing machinery. In addition, to determine the function of the C-terminal extension of the V protein, which is not translated in recent virulent field isolates, a translational stop codon was introduced to generate the corresponding short V protein. The mutant virus showed similar virus propagation and pathogenicity, indicating that C-terminal extension of the V protein is not relevant to virus pathogenesis. [Copyright &y& Elsevier]

TONS504 (C51H58O5I2) is a chlorin derivative that exhibits a photodynamic antimicrobial effect (PAE) on various infectious keratitis pathogens. However, the molecular characteristics of TONS504 are not well understood. This study aimed to investigate the molecular characteristics of TONS504 by comparing its singlet oxygen (1O 2) quantum yields and PAE with those of methylene blue (MB). To measure the 1O 2 quantum yields, TONS504 and MB were dissolved in phosphate-buffered saline and phosphate-buffered saline containing 1% Triton X-100. The solutions were then activated by a Nd:YAG laser with an average output power of 8 mW. Near-infrared 1O 2 luminescence was detected as an indicator of the 1O 2 quantum yields. To evaluate the PAE, TONS504 and MB were activated by a light-emitting diode with a total light energy of 30 J/cm2. We compared the minimum molar concentration of each photosensitizer to show apparent PAEs on Staphylococcus aureus , Pseudomonas aeruginosa , and Candida albicans. TONS504 exhibited higher 1O 2 quantum yields than MB in PBS/Triton X-100 but not in PBS. S. aureus and C. albicans were reduced by TONS504 at lower concentrations than by MB, but this was not the case for P. aeruginosa. Our results provide insight on the molecular characteristics of TONS504 and suggest that TONS504 has excellent 1O 2 quantum yields and PAE. Compared with MB, TONS504 in PBS has stronger efficacy toward some infectious keratitis pathogens but not others. • The efficacies of the photosensitizers TONS504 and methylene blue are compared. • TONS504 shows higher singlet oxygen quantum yields than methylene blue. • TONS504 shows photodynamic antimicrobial effects at lower concentrations than methylene blue. [ABSTRACT FROM AUTHOR]

The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferon regulatory factor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhibited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is important as an alternative target of paramyxovirus V proteins. [ABSTRACT FROM AUTHOR]

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding. [ABSTRACT FROM AUTHOR]