Culture supernatant fluid containing CVB4 was spiked (1% v/v) in medium containing BSA (50 mg mL1) (A) and in FBS (100%) (B), and 50 L of these suspensions were applied to petri dish lids in quadruplicate. These viral proteins can bind to receptors on the surface of adjoining cells, eliciting cell-cell fusion, and syncytia formation. proposed that 25HC altered cholesterol dynamics in a way that disrupted endosomal membrane physiology, limiting MRV particle trafficking and uncoating in the endosomes (Doms et al., 2018). Evolution of Viruses Although biologists have a significant amount of knowledge about how present-day viruses mutate and adapt, much less is known about how viruses originated in the first place. Copyright 2023 Wang, Zuo, Zhang, Bo, Zhang, Zhang and Wu. While information concerning CH25H in chickens is limited, there is an abundance of research available on other species. 316, 233250. Cholesterol-25-hydroxylase is a chicken ISG that restricts ALV-J infection by producing 25-hydroxycholesterol. 1998 Nov-Dec;56(6):693-703. Experiments were performed with CVB4 spiked (1% vol/vol final dilution) in FBS (100%) or water containing 50 mg mL1 BSA. Reovirus-induced tenosynovitis in chickens: the effect of breed. Taken together, these results indicated that ARV infection triggered the induction of CH25H at the early stage of infection. Microbiol. 1Universit Lille 2, Facult de Mdecine, CHRU Lille, Laboratoire de Virologie EA3610, Lille 59037, France, 2CHRU Lille Laboratoire de Bactriologie, Lille 59037, France. Avian Reovirus sigmaB interacts with Caveolin-1 in lipid rafts during dynamin-dependent Caveolae-mediated endocytosis. (G) The number of infectious virus particles was quantified by TCID50 analysis. doi: 10.1016/j.dci.2016.07.011, Raniga, K., and Liang, C. (2018). 2023 Jan 17;120(3):e2219962120. ARV has been implicated in several severe diseases and conditions that cause significant economic losses in the poultry industry, including viral arthritis/tenosynovitis, chronic respiratory disease, runting-stunting syndrome, and immunosuppression (Jones and Kibenge, 1984; Jones, 2000; Neelima et al., 2003). Our data emphasise the need for effective cleaning and disinfection in nosocomial settings to prevent pathogen transmission. CVB4 was more likely to be inactivated by drying in a protein-rich medium, whereas the impact of drying was reduced in the presence of sodium chloride. Rajtar B, Majek M, Polaski , Polz-Dacewicz M. Enteroviruses in water environmenta potential threat to public health. Biol. This is in contrast to viruses surrounded by a biological membrane, known as an envelope which contains lipids and proteins. Zhu et al. Samples were considered dried when liquid was no longer observed on the lids, which occurred within 2 h. The mean values of the infectious titers of droplets containing HSV-1, H1N1, and CVB4 were reduced by 2.33 log10; 1.1 log10; 1.5 log10, respectively, 2 h after the inoculation (Fig. Thereafter, dried inocula were recovered using 1 mL of medium and the infectious titers were determined and expressed as log10. and transmitted securely. The efficiency of recovering CVB4 when drops were dried in protein-rich media was analyzed. Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol. For example, after infection with viruses such as ZIKV, VSV and HIV, CH25H expression is significantly upregulated in host cells, whereas after infection with viruses such as PRRSV and HSV, CH25H expression is significantly downregulated. However, few studies characterized the association of non-enveloped viruses with CH25H and the role of CH25H in the regulation of ARV replication. The primer sequences for siRNA assay. Vet. Inocula on lids were dried for 2 h at room temperature, and, thereafter, recovered with 1 mL of culture media. Virus entry: looking Back and moving forward. Not all viruses have envelopes. Figure 3. B., Yutuc, E., Ma, X., Shen, S., Gomez Castro, M. F., et al. Thank you for visiting nature.com. . 19, 902912. Genomics and proteomics of vertebrate cholesterol ester lipase (LIPA) and cholesterol 25-hydroxylase (CH25H). Nat Microbiol 6, 13431344 (2021). Enveloped viruses are another class of potentially deadly viruses that we disinfect daily. 1. 6, 341363. Commun. The mRNA levels of the NS and C genes, NS protein levels, and ARV titers in the wild-type chCH25H group were significantly decreased, compared to the empty vector control group as expected. In addition, we revealed that 25HC produced by CH25H inhibits viral entry by delaying the kinetics of ARV uncoating, and CH25H blocks cellcell membrane fusion induced by the p10 protein of ARV. Microbiol. Figure 7. (2016). mBio 7:e01808-16. The results show that the chCH25H mutant (H242Q and H243Q) indeed loses the ability to produce 25HC (Supplementary Figure S3). Although entry is well understood for enveloped viruses1,2, less is known about the entry machinery of eukaryotic non-enveloped viruses. Lin Q, Lim JYC, Xue K, Yew PYM, Owh C, Chee PL, Loh XJ. The .gov means its official. Inhibition of pathogenic non-enveloped viruses by 25-hydroxycholesterol and 27-hydroxycholesterol. (2022). Detection of COVID-19 Virus on Surfaces Using Photonics: Challenges and Perspectives. doi: 10.1371/journal.ppat.1010322, Park, K., and Scott, A. L. (2010). CVB4 E2 is a strain provided by Ji-Won Yoon (Calgary, Alberta, Canada) (8, 13). Cholesterol 25-hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane cholesterol. A virus is made up of a DNA or RNA genome inside a protein shell called a capsid. The proteins in the envelope can include glycoproteins, which act as receptor . government site. Cell-cell fusion induced by reovirus FAST proteins enhances replication and pathogenicity of non-enveloped dsRNA viruses. In the second phase, the viral titers of each virus slowly decreased. The results are the mean + SD of four independent experiments. SiRNA1 and siRNA3 presented the most significant down-regulation of chCH25H (Supplementary Figure S2) and were subsequently chosen for interference experiments. Microbiol. doi: 10.1128/JVI.00767-19, Kusuma, A., Romero-Brey, I., Berger, C., Colpitts, C. C., Boldanova, T., Engelmann, M., et al. When cell lysis reached at least 75%, the inoculated flasks were scratched, freeze-thawed three times, and centrifuged at 2,000g for 10 min at 4C. The persistence of H1N1, HSV-1, MVM, and CVB4 was investigated and, for the first time, the effects of NaCl, FCS and BSA concentrations on CVB4 persistence were determined. doi: 10.1128/JVI.01047-18, Dong, H., Zhou, L., Ge, X., Guo, X., Han, J., and Yang, H. (2018). Microbiol. National Library of Medicine official website and that any information you provide is encrypted Besides that, a recent study using reverse genetics systems found that cellcell fusion mediated by FAST proteins enhances the replication and pathogenicity of non-enveloped dsRNA viruses (Kanai et al., 2019). doi: 10.20506/rst.19.2.1237, Jones, R. C., and Kibenge, F. S. (1984). As they are sensitive to several conditions, they didnt survive or reproduce when they are present in the gastrointestinal tract. Membranotropic peptides mediating viral entry. The mRNA expression levels of IFNA (A), IFNB (B), CH25H (C), ARV NS (D), and C (E) genes were determined by qRT-PCR at the indicated time points. Apart from antiviral mechanisms depending on enzyme activity, CH25H can also inhibit the infection of some viruses via a hydroxylase-independent mechanism (Ke et al., 2017; Lv et al., 2019; Li et al., 2020; Zhu et al., 2021). 79, 80908100. inactivated H1N1 at a higher temperature (28.3C vs. 20C) (2) and Abad et al. The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1178005/full#supplementary-material, Bauman, D. R., Bitmansour, A. D., McDonald, J. G., Thompson, B. M., Liang, G., and Russell, D. W. (2009). Furthermore, the addition of 25HC sufficiently complemented the lost activity of the chCH25H mutant in blocking syncytium formation (Figures 7B,C). The data are expressed as the meanstandard deviation. Biol. CH25H and 25HC have been shown to exhibit broad antiviral activity against a variety of viruses (Liu et al., 2013; Zhao et al., 2020). There was no significant difference in the number of syncytia between the mutant group and the empty vector group, while the relative number of syncytia in the mutant chCH25H group was significantly higher than those in the wild-type chCH25H group. Sane F, Caloone D, Gmyr V, Engelmann I, Belaich S, Kerr-Conte J, Pattou F, Desailloud R, Hober D. Coxsackievirus b4 can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of Pdx1 expression and disturbed formation of islet-like cell aggregates. Additionally, our previous study showed that cellular cholesterol in lipid rafts plays a critical role in ARV replication at both the entry stage and the post-entry stages (Wang Y. et al., 2020). Taken together, our data suggest that the most effective module for membrane disruption is a pentameric unit of full-length gamma, released from the virus, which associates with membranes via both N- and C-terminal ends. CVB4, which persisted for 5 weeks under the biosafety cabinet, was fully inactivated by 4 cycles of iterative drying, while H1N1 persisted for 5 d and remained infectious after 8 cycles. Unauthorized use of these marks is strictly prohibited. Survival and disinfection of parainfluenza viruses on environmental surfaces. KJ476699) was propagated and used as previously described (Duan et al., 2015; Wang Y. et al., 2020). Our studies suggest that full-length gamma, including the hydrophobic C terminus, forms an alpha-helical hairpin motif, and any disruption in this motif drastically reduces its functionality, in spite of the correct positioning of amphipathic 1 in the virus capsid. (2017). Google Scholar. Furthermore, it has been reported that HCV infection triggers the up-regulation of interferon-inducible CH25H in vivo, and 25HC primarily restricts HCV at the RNA replication level by inhibiting the formation of the viral replication factory (Kusuma et al., 2015). The virus is 100 times smaller than a single bacteria cell and is not alive; it only gets production after entering into the human or animal cell. Epub 2018 Feb 13. Polyinosinicpolycytidylic acid [poly (I:C)] was purchased from Sigma-Aldrich (St. Louis, United States) Wright-Giemsa Stain Kit was purchased from Baso Biotechnology (Zhuhai, China). 79, 85728580 (2005). In addition, when comparing the mutant group with the empty vector group, although the overexpression of mutant chCH25H increased the NS transcription and protein expression, the overexpression of mutant chCH25H had no significant effect on ARV C transcription and progeny virus production (Figure 5). 4). There are two types of viruses: enveloped and non-enveloped. Chicken CH25H inhibits ALV-J replication by promoting cellular autophagy. These results showed that iterative drying resulted in reductions in the viral titers of H1N1, CVB4, and HSV-1, whereas those of MVM remained unchanged. Tissues with resident macrophage populations (liver, lung, and brain) present the highest levels of CH25H induction (Bauman et al., 2009). (E) The mRNA expression levels of the ARV-C gene were determined by qRT-PCR. Oxysterols: an emerging class of broad spectrum antiviral effectors. Please enable it to take advantage of the complete set of features! Take the case of adenovirus, a non-enveloped virus, was demonstrated not to be affected by 25HC (Civra et al., 2014). HHS Vulnerability Disclosure, Help HD11 cells were infected with ARV at an MOI of 1. Viruses can also be classified by the design of their capsids. FOIA Structure of enveloped and non-enveloped viruses. The proteolysis kinetics of outer-capsid protein B were assessed both in the presence and absence of 25HC. Viruses 11:498. doi: 10.3390/v11060498, Xie, T., Feng, M., Zhang, X., Li, X., Mo, G., and Shi, M. (2023). This work was supported by the National Natural Science Foundation of China (31902250), the China Agriculture Research System of MOF and MARA (CARS-40), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). As shown in Figure 6B, no differences were observed between 25HC-treated (5M) and mock-treated ARV infection groups. J Hosp Infect. Non-enveloped virus lacks such covering and has capsid as the outermost part. We further tested whether the presence of 25HC restricts ARV replication after viral entry has taken place. Results are presented as means SD from three independent experiments. The efficiency of viral recovery from petri dish lids was confirmed in experiments combining the measurement of infectious titers and estimation of the levels of viral particles through the amount of viral RNA measured by RT real-time PCR. Further studies will be directed towards this line of investigation in our laboratory. In general, what distinguishes them is the presence (for enveloped viruses) or absence (for non-enveloped viruses) of a lipid bilayer membrane on the outer part of the virus. To investigate the suppressive effect of 25HC against ARV, the DF-1 cells were mock-treated or treated with the indicated concentrations (2.5, 5, and 10mM) of 25HC at the viral entry and post-entry stages. To further define the antiviral ability of CH25H against ARV, we knocked down the levels of the chCH25H in DF-1 cells using siRNA. 65, 218225. However, the detailed mechanisms should be evaluated in a future study. Ward RL, Ashley CS. The ARV NS protein levels of the mutant chCH25H were also much higher than those of the wild-type chCH25H group. Enveloped virus are sensitive to the conditions like drying and heating while non-enveloped virus even remains effective after drying and provides resistance against the conditions like heating and drying. cryo-electron microscopy; membrane lytic peptide; molecular dynamics simulation; non-enveloped virus; virus entry. Ashok D, Taheri M, Garg P, Webb D, Parajuli P, Wang Y, Funnell B, Taylor B, Tscharke DC, Tsuzuki T, Verma NK, Tricoli A, Nisbet DR. Adv Sci (Weinh). Immunol. The ARV replication levels in cells were evaluated using qRT-PCR and Western blotting at 24hpi. However, most plant viruses do not have a DNA genome; the majority have a +ssRNA genome, which acts like messenger RNA (mRNA). For these non-enveloped viruses, 25HC exerts antiviral activity by targeting members of oxysterol binding protein (OSBP) (e.g., Poliovirus, HRhV), targeting essential intracellular events of virus replication cycle (e.g., HPV-16), and sequestering viral particles into late endosomes (e.g., HRoV) (Civra et al., 2014, 2018; Roulin et al., 2014; Lembo et al., 2016; Doms et al., 2018). This study may provide new insights into the development of broad-spectrum antiviral therapies. RNA was extracted and the levels of viral RNA were measured by quantitative RT-PCR and expressed as Ct (). (2018). (F) The level of the ARV NS protein was analyzed by Western blotting. doi: 10.1016/j.mam.2016.04.003, Li, S., Li, L., Zhu, H., Shi, M., Fan, H., Gao, Y., et al. Some viruses have an external membrane envelope. Positive strand-specific RT was quantitated by the brilliant II QPCR kit (Agilent Technologies Stratagene) under universal cycle conditions (10 min at 95C, 40 cycles of 30 s at 60C) on a Mx3000p (Stratagene). *: P value <0.05. Hamilton MA, Russo RC, Thurston RV. Recently, CH25H and its product 25HC have been reported to exert multifaceted functions to regulate cholesterol homeostasis, antivirus process, inflammatory response, and cell survival (Cao et al., 2020; Zhao et al., 2020). In media containing low concentrations of NaCl from 0 to 0.1 mg mL1 the viral titers in the recovered dried spots increased slightly from 2.66 to 3.08 log10 TCID50 50 L1 (P=0.1). Cholesterol 25-hydroxylase expression following immune activation in response to SARS-CoV-2 infection. Department of Animal and Food ScienceUniversity of DelawareNon-enveloped viruses retain the FPson their Capsidand are still infectious Enveloped viruses lose the FPs on their Envelope, cannot bind to Receptor Molecules,and are no longer infectious Inclusion in an NLM database does not imply endorsement of, or agreement with, 39:e106057. Fifty microliters of clarified culture supernatant fluids from cells infected with CVB4, MVM, H1N1, or HSV-1 were applied to petri dish lid surfaces and then dried under the airflow of a class II biological safety cabinet at room temperature (20 2C). (2019). Table 2. The supernatant was aseptically aliquoted and stored in a 80C freezer. Would you like email updates of new search results? To investigate whether CH25H could inhibit ARV replication, we exogenously expressed myc-tagged chCH25H in DF-1 cells prior to ARV infection. Varicella zoster virus causes chicken pox and shingles. doi: 10.1189/jlb.0610318, Poynter, S. J., and DeWitte-Orr, S. J. doi: 10.1016/j.tim.2014.08.005, Civra, A., Cagno, V., Donalisio, M., Biasi, F., Leonarduzzi, G., Poli, G., et al. Before Internalized 25HC produced by CH25H accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion by blocking cholesterol export (Zang et al., 2020). Banerjee M, Khayat R, Walukiewicz HE, Odegard AL, Schneemann A, Johnson JE. It is responsible for around 200,000 deaths annually and outbreaks are common in small communities such as educational and care facilities. School of Clinical and Basic Medical Sciences, Shandong First Medical University and Shandong Academy of Medical Sciences, China, South China Agricultural University, China. The diameter of non-enveloped viruses with icosahedral symmetry, of various taxonomic groups (cauliflower mosaic virus, encephalomyocarditis virus, brome mosaic virus and bean mild mosaic virus), were measured. The dashed line represents the detection limit of the test. 19, 327336. doi: 10.15252/embj.2020106057, Wang, S., Wan, L., Ren, H., Xie, Z., Xie, L., Huang, J., et al. However, whether they can survive harsher . 2022 Aug;9(23):e2201415. Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion. Significance was determined by Students t-test (ns, not significant; *, p<0.05; **, p<0.01). Immunol. Survival of enteric viruses on environmental fomites. 2008 Feb;9(1):16-27. doi: 10.2174/138920308783565732. The trends were consistent with the transcription levels of the corresponding genes (Figures 1GI). pH modification of the effects of detergents on the stability of enteric viruses. 2020 Feb 1;1862(2):183102. doi: 10.1016/j.bbamem.2019.183102. Taha BA, Al Mashhadany Y, Bachok NN, Ashrif A Bakar A, Hafiz Mokhtar MH, Dzulkefly Bin Zan MS, Arsad N. Diagnostics (Basel). Accessibility (DF) The effects of 25HC treatment after ARV infection. These data indicated that overexpression of chCH25H significantly suppressed ARV infection. Furthermore, infectious titers higher than 106 TCID50 mL1 can be obtained for each virus in vitro. Virucidal effect of drying on CVB4 suspensions at various concentrations of FCS, BSA, and NaCl. The chicken CH25H gene (GenBank accession NM_001277354.1) was synthesized by GenScript (Nanjing) Co., Ltd. (Nanjing, China) and cloned into the pcDNA-3.1(+) vector (Invitrogen) to generate Myc-tagged expression plasmid pcDNA3.1-CH25H-Myc. In the present study, we preliminarily investigated the potential inhibition mechanism of 25HC against ARV at the viral entry stage. 19, 318330. Would you like email updates of new search results? To date, most literature describes the antiviral activities and the mechanism of CH25H against enveloped viruses, primarily by inhibiting virus-cell membrane fusion. and transmitted securely. In the absence of lipid envelopes and associated fusion proteins, non-enveloped viruses employ membrane lytic peptides to breach the limiting membranes of host cells. The chCH25H gene, located on chicken chromosome 6, is conserved across different species and is stimulated by interferons. The ARV strain GX/2010/1 (GenBank accession no. Inocula on lids were dried for 2 h at room temperature, and, thereafter, recovered with 1 mL of culture media. The stained cells were observed using an inverted microscope (Olympus, Tokyo, Japan), and the syncytium formation was determined by the average number of syncytia from five random fields using a10 objective. (2022). (B) The mRNA expression levels of the ARV-NS gene were determined by qRT-PCR. Beyond that, we found that 25HC treatment in DF1 cells significantly inhibits ARV replication in a dose-dependent manner at the entry and post-entry stages. Sci. 30, 34813500 (2011). We investigated the expression dynamics of endogenous CH25H after ARV infection in HD11 cells, which is a chicken macrophage cell line, considering that CH25H is an interferon-induced enzyme that belongs to the ISGs. The overexpression of chCH25H dramatically decreases ARV replication and the knockdown of chCH25H by siRNA promotes ARV infection. Virol. Results are presented as means SD from three independent experiments. Cholesterol 25-hydroxylase inhibits Senecavirus a replication by enzyme activity-dependent and independent mechanisms. Figure 1. reported that the first phase was characterized by water loss due to the evaporation of free water from the surface (14), which exposes viruses to a liquid-air interface, leading to virus inactivation (18). Unable to load your collection due to an error, Unable to load your delegates due to an error. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Annu. Print 2021 Apr. Unable to load your collection due to an error, Unable to load your delegates due to an error. RNA was extracted and the levels of viral RNA were measured by quantitative RT-PCR and expressed as Ct (). Mouse polyclonal antibodies against B and NS of ARV were prepared in our laboratory. The primer sequences for construction of the expression vector containing genes encoding Myc-tagged chCH25H mutant (H242Q and H243Q) by site-directed mutagenesis. At 36h post-transfection, the knockdown efficiency of CH25H was detected by qRT-PCR. In both protein-rich media, the amounts of viral RNA in recovered inocula dried for 2 h at room temperature and in undried inocula were similar, as displayed by the pattern of Ct values obtained by RT real-time PCR, whereas the infectious titer values were markedly different (Fig. doi: 10.1007/s00705-015-2415-1, Duncan, R. (2019). Epub 2023 Apr 25. This site needs JavaScript to work properly. Vet. These results revealed that the chCH25H mutant lost the ability to inhibit ARV replication. Avian reovirus (ARV) belongs to the Orthoreovirus genus of the Reoviridae family (Mertens, 2004). These viruses are less virulent as compared to the non-enveloped viruses, and are also sensitive to the conditions like drying and heating. Tissues with resident macrophage populations show the highest levels of CH25H induction (Bauman et al., 2009; Park and Scott, 2010). MeSH AIDS 34, 15931602. As expected, the siRNAs dramatically reduced chCH25H levels in DF-1 cells, and chCH25H RNA levels decreased by more than 85%, relative to that in cells transfected with NC siRNA (Figure 2D). Primers and probe pairs were designed with PrimerExpress software, and data were analyzed with Sequence Detector version 1.6.3 (both from Perkin- Elmer, Boston, Massachusetts). It has been reported that cholesterol 25-hydroxylase . Doms et al. Figure 5. In contrast, the viral titers in media containing between 0.1 and 2 mg mL1 NaCl decreased from 3.08 to 2.58 log10 TCID50 50 L1 (P=0.04).