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Clinical data from over two decades, involving more than 3000 treated patients, demonstrate that adeno‐associated virus (AAV) gene therapy is a safe, effective, and well‐tolerated therapeutic method. Clinical trials using AAV‐mediated gene delivery to accessible tissues have led to successful treatments for numerous monogenic disorders and advancements in tissue engineering. Although the US Food and Drug Administration (FDA) has approved AAV for clinical use, systemic administration remains a significant challenge. In this review, we delve into AAV biology, focusing on current manufacturing technologies and transgene engineering strategies. We examine the use of AAVs in ongoing clinical trials for ocular, neurological, and hematological disorders, as well as cancers. By discussing recent advancements and current challenges in the field, we aim to provide valuable insights for researchers and clinicians navigating the evolving landscape of AAV‐based gene therapy. [ABSTRACT FROM AUTHOR]

Abstract In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture.

In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture. [ABSTRACT FROM AUTHOR]

Abstract Although we have gained insight into the biological and biochemical effects of natural sunlight exposure on prokaryotes, little is known about sunlight exposure on natural virus communities. To address this question, an investigation of the effects of sunlight and dark treatments on viral communities, viral production and decay rates in Kenting coastal waters was conducted. The average rate of net viral production in the sunlight and dark treatment was 0.010 and 0.018 × 106 viruses mL–1 h− 1, respectively. Furthermore, averaged value for viral decay in the sunlight treatment was 0.016 × 106 viruses mL− 1 h− 1, a significant decrease (ca. 60%=((0.83 − 0.33)/(0.83 × 100%)) was observed in sunlight conditions, whereas no significant changes occurred in dark conditions. The gross viral production under sunlight conditions was slightly higher, however, non-significantly higher than that in the dark treatment. As a result, we suggest that sunlight damages a large portion of the natural viral community, affecting the role viruses play in food webs.

Although we have gained insight into the biological and biochemical effects of natural sunlight exposure on prokaryotes, little is known about sunlight exposure on natural virus communities. To address this question, an investigation of the effects of sunlight and dark treatments on viral communities, viral production and decay rates in Kenting coastal waters was conducted. The average rate of net viral production in the sunlight and dark treatment was 0.010 and 0.018 × 106 viruses mL–1 h− 1, respectively. Furthermore, averaged value for viral decay in the sunlight treatment was 0.016 × 106 viruses mL− 1 h− 1, a significant decrease (ca. 60%=((0.83 − 0.33)/(0.83 × 100%)) was observed in sunlight conditions, whereas no significant changes occurred in dark conditions. The gross viral production under sunlight conditions was slightly higher, however, non-significantly higher than that in the dark treatment. As a result, we suggest that sunlight damages a large portion of the natural viral community, affecting the role viruses play in food webs. [ABSTRACT FROM AUTHOR]

Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent in vitro and in vivo data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.

The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness. [ABSTRACT FROM AUTHOR]

Viral dynamics are the result of the balance between the rates of viral production and decay. Here, we have carried out independent measurements of viral production and decay rates in different depths of the southern East China Sea in summer (August and October 2021). In this study, the prevalence of viral abundance at the surface waters (14.2~27.6 × 105 viruses mL−1) was significantly higher than the bottom of the euphotic zone (2.9~12.6 × 105 viruses mL−1). As for viruses to bacteria ratio (VBR) values, we found a wide variability both at the surface (1.4 to 3.2) and bottom of the euphotic zone (2.1 to 16.2). The results of our study showed that at all stations examined, in the southern East China Sea, the values of gross viral production (GVP) were significantly higher in the sunlit surfaces compared to the bottom of the euphotic zone. In particular, our analysis indicates that no significant viral decay rates (VD) were observed in some regions at the bottom of the euphotic zone. Here, we also provide a budget for viral abundance and net viral production in different regions in the southern East China Sea. The GVP or VD is not applicable in our case to explain VBR is high at bottom of the euphotic zone. The mechanisms underlying VBR uncoupling, viral production, and viral loss in marine systems are still being investigated. [ABSTRACT FROM AUTHOR]

Although we have gained insight into the biological and biochemical effects of natural sunlight exposure on prokaryotes, little is known about sunlight exposure on natural virus communities. To address this question, an investigation of the effects of sunlight and dark treatments on viral communities, viral production and decay rates in Kenting coastal waters was conducted. The average rate of net viral production in the sunlight and dark treatment was 0.010 and 0.018 x 106 viruses mL-1 h-1, respectively. Furthermore, averaged value for viral decay in the sunlight treatment was 0.016 x 106 viruses mL-1 h-1, a significant decrease (ca. 60%=((0.83 - 0.33)/(0.83 x 100%)) was observed in sunlight conditions, whereas no significant changes occurred in dark conditions. The gross viral production under sunlight conditions was slightly higher, however, non-significantly higher than that in the dark treatment. As a result, we suggest that sunlight damages a large portion of the natural viral community, affecting the role viruses play in food webs. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five FDA-approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield., Competing Interests: DECLARATION OF INTERESTS: B.L.D. serves on the advisory board of Latus Biosciences, Patch Bio, Spirovant Biosciences, Resilience, and Carbon Biosciences and has sponsored research unrelated to this work from Roche, Latus, and Spirovant. Authors have filed a patent related to this manuscript through the Children’s Hospital of Philadelphia.

The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness.

Viral dynamics are the result of the balance between the rates of viral production and decay. Here, we have carried out independent measurements of viral production and decay rates in different depths of the southern East China Sea in summer (August and October 2021). In this study, the prevalence of viral abundance at the surface waters (14.2~27.6 × 105 viruses mL−1) was significantly higher than the bottom of the euphotic zone (2.9~12.6 × 105 viruses mL−1). As for viruses to bacteria ratio (VBR) values, we found a wide variability both at the surface (1.4 to 3.2) and bottom of the euphotic zone (2.1 to 16.2). The results of our study showed that at all stations examined, in the southern East China Sea, the values of gross viral production (GVP) were significantly higher in the sunlit surfaces compared to the bottom of the euphotic zone. In particular, our analysis indicates that no significant viral decay rates (VD) were observed in some regions at the bottom of the euphotic zone. Here, we also provide a budget for viral abundance and net viral production in different regions in the southern East China Sea. The GVP or VD is not applicable in our case to explain VBR is high at bottom of the euphotic zone. The mechanisms underlying VBR uncoupling, viral production, and viral loss in marine systems are still being investigated.

In spite of the fact that the interactions between environmental parameters and prokaryotic and viral abundance have been explored in various aquatic environments, only a few independent estimates of viral production and decay in the estuarine region have been explored. In this study, data were analyzed for viral and prokaryotic abundance, viral production, and viral decay in a subtropical Danshui estuary in summer 2021. Prokaryotic abundance varied from 2.4 ± 0.6 × 105 to 12 ± 2.3 × 105 cells mL−1, and viral abundance ranged from 2.3 ± 0.9 × 105 to 6.9 ± 1.3 × 105 viruses mL−1 during the study period. Viral abundance was significantly correlated with prokaryotic abundance and chlorophyll a concentration. Furthermore, studies of changes in viral to prokaryotic abundance ratio (VPR) ranged from 0.42 ± 0.11 to 2.0 ± 0.25. Viral decay values were 2.1 ± 0.5 and 2.1 ± 0.3 × 104 virus mL−1h−1, and non-significant differences were observed between the inner estuary and coastal water region. Viral decay almost balanced gross viral production in this study. The dilution experiments revealed non-significant net viral production in July; thus, a lower VPR might be explained in this estuarine environment. [ABSTRACT FROM AUTHOR]

In freshwater environments, limited data exist on the impact of mortality forces (viruses and heterotrophic nanoflagellates) on bacterial growth efficiency (BGE, index of bacterial carbon metabolism) compared to resource availability. An investigation to determine the relative influence of viral lysis and flagellate predation (top-down forces) on BGE was conducted in a mesotrophic freshwater system (Lake Goule, France) with time and space. Viral abundance was significantly (p < 0.001) related to bacterial abundance by a power law function with an exponent less than 1, emphasizing that the increases in host population (bacteria) together with viruses were not proportionate. A lytic viral strategy was evident throughout the study period, with high lysis of the bacterial population (up to 60%) supported by viral production rates. Viral processes (lysis and production) that were influenced by bacterial production and heterotrophic nanoflagellate abundance had a positive impact on BGE. Estimates of BGE were variable (9.9–45.5%) due to uncoupling between two metabolic parameters—namely bacterial production and respiration. The existence of a synergistic relationship between viruses and flagellates with bacteria in Lake Goule highlighted the decisive impact of top-down agents in sustaining the bacterial carbon metabolism of non-infected population through the nature of vital resources released via mortality processes. [ABSTRACT FROM AUTHOR]

Viral processes in the coastal and offshore regions of the Eastern Arabian Sea (west coast of India) and their contribution of viral lysis to the organic carbon and nitrogen pool was examined. Water samples were collected during the southwest monsoon season at different depths (up to 1000 m) from four transects, with each transect consisting of two sampling points (S1–S8). Abundances of viruses and prokaryotes together with viral mediated prokaryotic mortality (up to 49.7%) were significantly (p < 0.001) higher in eutrophic coastal stations, whereas high percent lysogeny (up to 93%) was observed in the offshore regions. High viral-mediated carbon (Mean ± SD = 67.47 ± 2.0 μM C L−1 d−1) and nitrogen (Mean ± SD = 13.49 ± 14.0 μM N L−1 d−1) release was evident in the surface coastal waters compared to offshore regions. The percentage contributions of carbon and nitrogen released by viral lysis to the total dissolved organic carbon and nitrogen pool were estimated to be 7.4% and 3.9%, respectively, in the coastal surface waters. Our findings suggest that the contribution of viral lysis to DOM production through viral shunt could be crucial for the cycling of major biogeochemical elements and functioning of the studied tropical ecosystem. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production. [ABSTRACT FROM AUTHOR]

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-μm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-μm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-μm filters and diluted with virus-free seawater. Viral production in the <0.2-μm filtrate was estimated from changes in viral abundance determined through flow cytometry. We found that viruses were produced in the <0.2-μm communities, which were strongly enriched with low nucleic acid content bacteria. Estimated viral production in the <0.2-μm filtrates accounted for up to 43% of total viral production and 10% of dissolved organic carbon production mediated by viral lysis of bacterial cells. By not considering viral production in these <0.2-μm communities, the virus reduction assay may underestimate viral production. Virus–bacteria interactions in <0.2-μm communities may represent a significant and overlooked role of viruses in marine food webs and carbon fluxes. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-μm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-μm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-μm filters and diluted with virus-free seawater. Viral production in the

[Display omitted] A key to tackling the coronavirus disease 2019 (COVID-19) pandemic is to understand how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manages to outsmart host antiviral defense mechanisms. Stress granules (SGs), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. Here, we show that the SARS-CoV-2 nucleocapsid (N) protein, an RNA binding protein essential for viral production, interacted with Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) and disrupted SG assembly, both of which require intrinsically disordered region1 (IDR1) in N protein. The N protein partitioned into SGs through liquid-liquid phase separation with G3BP, and blocked the interaction of G3BP1 with other SG-related proteins. Moreover, the N protein domains important for phase separation with G3BP and SG disassembly were required for SARS-CoV-2 viral production. We propose that N protein-mediated SG disassembly is crucial for SARS-CoV-2 production. [ABSTRACT FROM AUTHOR]

Viruses are key players in the marine ecosystem. It is critical to study specific viral processes and their inter-relationship with various biotic and abiotic variables to quantify their impact on the marine environment. This study investigates the influence of seasonality on viral distribution and their mediated processes in the coastal region of the southeastern Arabian Sea (India) for two consecutive years (2014 and 2015). Water samples were collected from four sampling stations, S1, S2, S3 and S4, in the southeastern Arabian Sea on a monthly basis. Samples were analyzed for the variations in viral abundance, viral production, and viral-induced host mortality covering three seasons namely, pre-monsoon, monsoon and post-monsoon. Viruses and bacteria were enumerated by epifluorescence microscopy following staining with SYBR Green I and viral production was estimated using viral dilution method. Seasonal variations in viral-mediated mortality and microzooplankton grazing mortality of phytoplankton hosts were estimated by modified dilution method. The results revealed a pronounced seasonal pattern of viral abundance (0.04–4.03 × 107 viruses mL−1) and viral production (0.71 × 1010–4.94 × 1010 L−1d−1). The observed high viral-induced mortality of prokaryotes (25.85%) and phytoplankton (7.98–28.9%) during the pre-monsoon season was eventually linked to high viral abundance and production rates. Viral-mediated processes were essentially linked to host density and water temperature. Transmission electron microscopy analysis revealed that myoviruses and non-tailed viruses were dominant in the study region with rod-shaped bacteria being more susceptible to viral infection. The results indicated the prevalence of seasonally induced active viral processes in this tropical marine ecosystem which may significantly contribute to rapid recycling of nutrients in this region. [ABSTRACT FROM AUTHOR]

In freshwater environments, limited data exist on the impact of mortality forces (viruses and heterotrophic nanoflagellates) on bacterial growth efficiency (BGE, index of bacterial carbon metabolism) compared to resource availability. An investigation to determine the relative influence of viral lysis and flagellate predation (top-down forces) on BGE was conducted in a mesotrophic freshwater system (Lake Goule, France) with time and space. Viral abundance was significantly (p < 0.001) related to bacterial abundance by a power law function with an exponent less than 1, emphasizing that the increases in host population (bacteria) together with viruses were not proportionate. A lytic viral strategy was evident throughout the study period, with high lysis of the bacterial population (up to 60%) supported by viral production rates. Viral processes (lysis and production) that were influenced by bacterial production and heterotrophic nanoflagellate abundance had a positive impact on BGE. Estimates of BGE were variable (9.9–45.5%) due to uncoupling between two metabolic parameters—namely bacterial production and respiration. The existence of a synergistic relationship between viruses and flagellates with bacteria in Lake Goule highlighted the decisive impact of top-down agents in sustaining the bacterial carbon metabolism of non-infected population through the nature of vital resources released via mortality processes.

Viral processes in the coastal and offshore regions of the Eastern Arabian Sea (west coast of India) and their contribution of viral lysis to the organic carbon and nitrogen pool was examined. Water samples were collected during the southwest monsoon season at different depths (up to 1000 m) from four transects, with each transect consisting of two sampling points (S1–S8). Abundances of viruses and prokaryotes together with viral mediated prokaryotic mortality (up to 49.7%) were significantly (p < 0.001) higher in eutrophic coastal stations, whereas high percent lysogeny (up to 93%) was observed in the offshore regions. High viral-mediated carbon (Mean ± SD = 67.47 ± 2.0 μM C L−1 d−1) and nitrogen (Mean ± SD = 13.49 ± 14.0 μM N L−1 d−1) release was evident in the surface coastal waters compared to offshore regions. The percentage contributions of carbon and nitrogen released by viral lysis to the total dissolved organic carbon and nitrogen pool were estimated to be 7.4% and 3.9%, respectively, in the coastal surface waters. Our findings suggest that the contribution of viral lysis to DOM production through viral shunt could be crucial for the cycling of major biogeochemical elements and functioning of the studied tropical ecosystem.

In spite of the fact that the interactions between environmental parameters and prokaryotic and viral abundance have been explored in various aquatic environments, only a few independent estimates of viral production and decay in the estuarine region have been explored. In this study, data were analyzed for viral and prokaryotic abundance, viral production, and viral decay in a subtropical Danshui estuary in summer 2021. Prokaryotic abundance varied from 2.4 ± 0.6 × 105 to 12 ± 2.3 × 105 cells mL−1, and viral abundance ranged from 2.3 ± 0.9 × 105 to 6.9 ± 1.3 × 105 viruses mL−1 during the study period. Viral abundance was significantly correlated with prokaryotic abundance and chlorophyll a concentration. Furthermore, studies of changes in viral to prokaryotic abundance ratio (VPR) ranged from 0.42 ± 0.11 to 2.0 ± 0.25. Viral decay values were 2.1 ± 0.5 and 2.1 ± 0.3 × 104 virus mL−1h−1, and non-significant differences were observed between the inner estuary and coastal water region. Viral decay almost balanced gross viral production in this study. The dilution experiments revealed non-significant net viral production in July; thus, a lower VPR might be explained in this estuarine environment.

Hand, foot, and mouth disease (HFMD) is a febrile exanthematous disease with typical or atypical symptoms. Typical HFMD is usually caused by enterovirus 71 (EV71) or coxsackievirus A16, while atypical HFMD is usually caused by coxsackievirus A6 (CA6). In recent years, worldwide outbreaks of CA6-associated HFMD have dramatically increased, although the pathogenic mechanism of CA6 is still unclear. EV71 has been established to induce caspase-dependent apoptosis, but in this study, we demonstrate that CA6 infection promotes a distinct pathway of cell death that involves loss of cell membrane integrity. Necrostatin-1, an inhibitor of necroptosis, blocks the cell death induced by CA6 infection, but Z-DEVD-FMK, an inhibitor of caspase-3, has no effect on CA6-induced cell death. Furthermore, CA6 infection up-regulates the expression of the necroptosis signaling molecule RIPK3. Importantly, necrostatin-1 inhibits CA6 viral production, as assessed by its ability to inhibit levels of VP1 protein and genomic RNA and infectious particles. CA6-induced necroptosis is not dependent on the generation of reactive oxygen species; however, viral 3D protein can directly bind RIPK3, which is suggestive of a direct mechanism of necroptosis induction. Therefore, these results indicate that CA6 induces a mechanism of RIPK3-dependent necroptosis for viral production that is distinct from the mechanism of apoptosis induced by typical HFMD viruses.

Enterovirus D68 (EVD68) is an emerging pathogen that recently caused a large worldwide outbreak of severe respiratory disease in children. However, the relationship between EVD68 and host cells remains unclear. Caspases are involved in cell death, immune response, and even viral production. We found that caspase-3 was activated during EVD68 replication to induce apoptosis. Caspase-3 inhibitor (Z-DEVD-FMK) inhibited viral production, protected host cells from the cytopathic effects of EVD68 infection, and prevented EVD68 from regulating the host cell cycle at G0/G1. Meanwhile, caspase-3 activator (PAC-1) increased EVD68 production. EVD68 infection therefore activates caspase-3 for virus production. This knowledge provides a potential direction for the prevention and treatment of disease related to EVD68. [ABSTRACT FROM AUTHOR]

The Zika virus (ZIKV) is a mosquito-borne flavivirus that has reemerged as a serious public health problem around the world. Syndromes of infected people range from asymptomatic infections to severe neurological disorders, such as Guillain-Barré syndrome and microcephaly. Screening anti-ZIKV drugs derived from Chinese medicinal herbs is one method of identifying antiviral agents. In this paper, we report that (1) Cephalotaxine (CET), an alkaloid isolated from Cephalotaxus drupacea , was effective in inhibiting ZIKV activity in vitro (i.e., in Vero and A549 cell lines) and (2) the mechanisms which underlie these effects involve virucidal activity and a decrease in viral replication. Specifically, CET was found to decrease ZIKV RNA and viral protein expression, inhibit ZIKV replication, and inhibit ZIKV mRNA/protein production. We also determined that CET is effective in inhibiting dengue virus 1–4 (DENV1-4). Taken together, our findings indicate that CET could be an effective lead compound in the treatment of ZIKV and also suggest that further investigation and development of CET-derived drugs may lead to a new class of anti- Flavivirus medications. • CET against ZIKV infection via inhibiting replication and stability. • CET was identified as a potent inhibitor of ZIKV infection. • CET as a candidate compound for potential ZIKV treatment. [ABSTRACT FROM AUTHOR]

Hand, foot, and mouth disease (HFMD) is a febrile exanthematous disease with typical or atypical symptoms. Typical HFMD is usually caused by enterovirus 71 (EV71) or coxsackievirus A16, while atypical HFMD is usually caused by coxsackievirus A6 (CA6). In recent years, worldwide outbreaks of CA6-associated HFMD have dramatically increased, although the pathogenic mechanism of CA6 is still unclear. EV71 has been established to induce caspase-dependent apoptosis, but in this study, we demonstrate that CA6 infection promotes a distinct pathway of cell death that involves loss of cell membrane integrity. Necrostatin-1, an inhibitor of necroptosis, blocks the cell death induced by CA6 infection, but Z-DEVD-FMK, an inhibitor of caspase-3, has no effect on CA6-induced cell death. Furthermore, CA6 infection up-regulates the expression of the necroptosis signaling molecule RIPK3. Importantly, necrostatin-1 inhibits CA6 viral production, as assessed by its ability to inhibit levels of VP1 protein and genomic RNA and infectious particles. CA6-induced necroptosis is not dependent on the generation of reactive oxygen species; however, viral 3D protein can directly bind RIPK3, which is suggestive of a direct mechanism of necroptosis induction. Therefore, these results indicate that CA6 induces a mechanism of RIPK3-dependent necroptosis for viral production that is distinct from the mechanism of apoptosis induced by typical HFMD viruses. [ABSTRACT FROM AUTHOR]

Virioplankton is an important component of the aquatic ecosystem and plays multiple ecological and biogeochemical roles. Although the spatial and temporal distributions and dynamics of virioplankton have been well investigated in riverine and marine environments, little is known about the dynamics and environmental controlling mechanisms of virioplankton in estuaries. In this study, viral abundance, production and decay were examined in the Pearl River Estuary (PRE), one of the largest estuaries in China. The influences of freshwater and seawater mixing on viral ecological dynamics were evaluated with several cross-transplant experiments. In PRE, viral abundance, production and decay rates varied from 2.72 ± 0.09 to 27.5 ± 1.07 × 106 viruses ml−1, 7.98 ± 2.33 to 16.27 ± 2.85% h−1 and 0.80 ± 0.23 to 3.74 ± 0.98% h−1, respectively. When the riverine and marine microbial community were transferred into simulated brackish water, viral production rates were markedly inhibited by 83.8% and 47.3%, respectively. The decay of riverine and marine virioplankton was inhibited by 21.1% and 34.2%, respectively, in simulated brackish water. These results indicate change of estuarine environmental factors significantly alters the dynamics of riverine and marine virioplankton. In addition, the effects of mixing on viral production and decay differed between high- and low-fluorescence viruses. High-fluorescence viruses seemed more resistant to decay than low-fluorescence viruses, whereas the production of marine low-fluorescence viruses seemed more resistant to inhibition than that of marine high-fluorescence viruses. Together, these results provide new insights into the ecological dynamics of virioplankton in estuarine environments. [ABSTRACT FROM AUTHOR]

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Viral production (VP) and bacterial mortality by viral lysis critically influence the production and mortality of aquatic bacteria. Although bacterial production, mortality by viral lysis, and viral density have been found to exhibit diel variations, the diel change in viral production has rarely been investigated. In this study, we conducted two diel dilution incubation experiments in a semi-enclosed, nutrient-rich coastal region in northeastern Taiwan to estimate the diel viral production and the mortality by viral lysis. We also compared two methods (linear regression between viral density and time versus arithmetic mean of VP during incubation) of estimating viral production. We found that viral production estimated by linear regression and bacterial mortality by viral lysis were higher during the daytime than during the nighttime. A possible explanation for the high viral production at daytime is that the bacterial community was composed of cell types with higher burst sizes at daytime. We further argued that the classical linear regression method can be used only when viral density significantly linearly increases with time, which does not always occur in dilution incubations. This study offered observations of diel variation in viral dynamics and discussed the methods estimating viral production in a marine environment.

Climate warming can directly affect biological processes in marine environments. Here, we investigated if warming (+2 °C) can change dynamics in viral and prokaryotic populations in the cold seasons in natural seawaters. We monitored the changes in viral production and prokaryotic growth rate. The prokaryotic average gross growth rates were 0.08 and 0.34 h−1 in November and 0.06 and 0.41 h−1 in December in the in situ and warming experiments, respectively. We found that warming water temperature resulted in a significant increase in prokaryotic growth rates. In warming experiments, the overall viral production rate was about 0.77–14.4 × 105 viruses mL−1 h−1, and a rough estimate of prokaryotic mortality was about 5.6–6.8 × 104 cells mL−1 h−1. Based on our estimation, burst sizes of about 21 and 14 viruses prokaryotes−1 were measured under the experimental warming period. Moreover, the results found that an increased water temperature in the subtropical western Pacific coastal waters increases prokaryotic growth rates, enhances viral production, and changes the carbon fluxes in the trophic interactions of microbes.

Viruses are the most abundant microorganisms in marine environments and viral infections can be either lytic (virulent) or lysogenic (temperate phage) within the host cell. The aim of this study was to quantify viral dynamics (abundance and infection) in the coastal Red Sea, a narrow oligotrophic basin with high surface water temperatures (22–32 °C degrees), high salinity (37.5–41) and continuous high insolation, thus making it a stable and relatively unexplored environment. We quantified viral and environmental changes in the Red Sea (two years) and the occurrence of lysogenic bacteria (induced by mitomycin C) on the second year. Water temperatures ranged from 24.0 to 32.5 °C, and total viral and bacterial abundances ranged from 1.5 to 8.7 × 106 viruses mL−1 and 1.9 to 3.2 × 105 bacteria mL−1, respectively. On average, 12.24% ± 4.8 (SE) of the prophage bacteria could be induced by mitomycin C, with the highest percentage of 55.8% observed in January 2018 when bacterial abundances were low; whereas no induction was measurable in spring when bacterial abundances were highest. Thus, despite the fact that the Red Sea might be perceived as stable, warm and saline, relatively modest changes in seasonal conditions were associated with large swings in the prevalence of lysogeny.

HIV Nef is a central auxiliary protein in HIV infection and pathogenesis. Our results indicate that HDAC6 promotes the aggresome/autophagic degradation of the viral polyprotein Pr55Gag to inhibit HIV-1 production. Nef counteracts this antiviral activity of HDAC6 by inducing its degradation and subsequently stabilizing Pr55Gag and Vif viral proteins. Nef appears to neutralize HDAC6 by an acidic/endosomal-lysosomal processing and does not need the downregulation function, since data obtained with the non-associated cell-surface Nef-G2A mutant – the cytoplasmic location of HDAC6 – together with studies with chemical inhibitors and other Nef mutants, point to this direction. Hence, the polyproline rich region P72xxP75 (69–77 aa) and the di-Leucin motif in the Nef-ExxxLL160-165 sequence of Nef, appear to be responsible for HDAC6 clearance and, therefore, required for this novel Nef proviral function. Nef and Nef-G2A co-immunoprecipitate with HDAC6, whereas the Nef-PPAA mutant showed a reduced interaction with the anti-HIV-1 enzyme. Thus, the P72xxP75 motif appears to be responsible, directly or indirectly, for the interaction of Nef with HDAC6. Remarkably, by neutralizing HDAC6, Nef assures Pr55Gag location and aggregation at plasma membrane, as observed by TIRFM, promotes viral egress, and enhances the infectivity of viral particles. Consequently, our results suggest that HDAC6 acts as an anti-HIV-1 restriction factor, limiting viral production and infection by targeting Pr55Gag and Vif. This function is counteracted by functional HIV-1 Nef, in order to assure viral production and infection capacities. The interplay between HIV-1 Nef and cellular HDAC6 may determine viral infection and pathogenesis, representing both molecules as key targets to battling HIV. [ABSTRACT FROM AUTHOR]

Human enterovirus 68 (EVD68) is a primary causative agent for respiratory illness worldwide. Until now, there has been no available medication for treating EVD68-related diseases. Rheum emodin, artemisinin, astragaloside, pseudolaric acid B, oridonin, and erianin are natural extracts from Chinese herbs that have traditionally been used for the treatment and prevention of epidemic diseases. Our results showed that pseudolaric acid B protected cells from EVD68-induced cytopathic effects and decreased viral production. However, the same effects were not observed with rheum emodin, astragaloside, or artemisinin. Pseudolaric acid B inhibited EVD68 production by manipulating the host cell cycle in G2/M phase. Further, either oridonin or erianin related G2/M arrest also inhibited viral production. Due to inducing G2/M phase arrest, pseudolaric acid B, oridonin, and erianin might be good candidates for inhibiting EVD68 production, and Chinese herbs with natural compounds inducing G2/M arrest should be considered for the treatment of EVD68-related diseases. [ABSTRACT FROM AUTHOR]

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

Objective of the study is to investigate the sensitivity of different animals to highly pathogenic Orthopoxviruses applying techniques, based on utilization of primary cultures of lung cells, and to assess the possibility of further deployment of this approach. Materials and methods. Cultural and virological research methods are used. Results and conclusions. Performed is the assessment of sensitivity of outbred mice, marmots and chickens to variola virus (VV) and monkeypox virus (MPV), using suspended primary cultures of lung cells (SPCLC) of these animals. Through inoculation of the mentioned above cell cultures with VV and MPV in a dose of 0.00001 PFU per a cell (plaque forming unit /cell) demonstrated has been virus replication with maximum concentration values in all cases (1,4 - 2,0 lg PFU/ml), mainly 3 days after infection. According to the data on SPCLC, sensitivity to VV in mice, marmots and chickens (ID50 - 50 % infective dose) amounts to (1,3 ± 0,5) lg PFU; (2,3 ± 0,5) lg PFU; and (0,0 ± 0,4) lg PFU respectively, taking into account unhindered interaction of the virus with permissive lung cells in the organism of the animals. As for MPV values for this indicator, they are: (1,7 ± 0,3) lg PFU for mice, and (0,5 ± 0,3) lg PFU - for marmots. Obtained ID50 values for VV using mice SPCLC and for MPV using mice and marmots SPCLC coincide with the ones, studied in direct experiments on intranasal infection with the viruses, with regard to 10 % of the viral application in lungs when deploying the latter method of infection. The fact testifies to the possibility of further deployment of this method for the assessment of animal sensitivity to highly pathogenic Orthopoxviruses based on the results of in vitro experiments.

The lytic and lysogenic life cycles of marine phages are influenced by environmental conditions such as solar radiation, temperature, and host abundance. Temperature can regulate phage infection, but its role is difficult to discern in oligotrophic waters where there is typically low host abundance and high temperatures. Here, we study the temporal variability of viral dynamics and the occurrence of lysogeny using mitomycin C in a eutrophic coastal lagoon in the oligotrophic Red Sea, which showed strong seasonality in terms of temperature (22.1–33.3 °C) and large phytoplankton blooms. Viral abundances ranged from 2.2 × 106 to 1.5 × 107 viruses mL−1 and were closely related to chlorophyll a (chl a) concentration. Observed high virus-to-bacterium ratio (VBR) (4–79; 16 ± 4 (SE)) suggests that phages exerted a tight control of their hosts as indicated by the significant decrease in bacterial abundance with increasing virus concentration. Heterotrophic bacterial abundance also showed a significant decrease with increasing temperature. However, viral abundance was not related to temperature changes and the interaction of water temperature, suggesting an indirect effect of temperature on decreased host abundance, which was observed at the end of the summertime. From the estimated burst size (BS), we observed lysogeny (undetectable to 29.1%) at low percentages of 5.0% ± 1.2 (SE) in half of the incubations with mitomycin C, while it increased to 23.9% ± 2.8 (SE) when the host abundance decreased. The results suggest that lytic phages predominate, switching to a moderate proportion of temperate phages when the host abundance reduces.

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production.

Previous studies demonstrate that human enterovirus 71 (EV71), a primary causative agent for hand, foot, and mouth disease, activates caspase-3 through the non-structural viral 3C protein to induce host cell apoptosis; however, until now it was unclear how 3C activates caspase-3 and how caspase-3 activation affects viral production. Our results demonstrate that 3C binds caspase-8 and caspase-9 but does not directly bind caspase-3 to activate them, and that the proteolytic activity of 3C is required by the activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity attenuates apoptosis in 3C-transfected cells. Furthermore, caspase-3 inhibitor protects host cells from the cytopathic effect of EV71 infection and prevents cell cycle arrest, which is known to be favored for EV71 viral replication. Inhibition of caspase-3 activity decreases EV71 viral protein expression and viral production, but has no effect on viral entry, replication, even polyprotein translation. Therefore, caspase-3 is exploited functionally by EV71 to facilitate its production, which suggests a novel therapeutic approach for the treatment and prevention of hand, foot, and mouth disease.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment., Competing Interests: The authors declare no conflict of interest.

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production. [ABSTRACT FROM AUTHOR]

Previous studies demonstrate that human enterovirus 71 (EV71), a primary causative agent for hand, foot, and mouth disease, activates caspase-3 through the non-structural viral 3C protein to induce host cell apoptosis; however, until now it was unclear how 3C activates caspase-3 and how caspase-3 activation affects viral production. Our results demonstrate that 3C binds caspase-8 and caspase-9 but does not directly bind caspase-3 to activate them, and that the proteolytic activity of 3C is required by the activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity attenuates apoptosis in 3C-transfected cells. Furthermore, caspase-3 inhibitor protects host cells from the cytopathic effect of EV71 infection and prevents cell cycle arrest, which is known to be favored for EV71 viral replication. Inhibition of caspase-3 activity decreases EV71 viral protein expression and viral production, but has no effect on viral entry, replication, even polyprotein translation. Therefore, caspase-3 is exploited functionally by EV71 to facilitate its production, which suggests a novel therapeutic approach for the treatment and prevention of hand, foot, and mouth disease. [ABSTRACT FROM AUTHOR]

A new robust adaptive controller is developed for the control of the hepatitis B virus (HBV) infection inside the body. The non‐linear HBV model has three state variables: uninfected cells, infected cells and free viruses. A control law is designed for the antiviral therapy such that the volume of infected cells and the volume of free viruses are decreased to their desired values which are zero. One control input represents the efficiency of drug therapy in inhibiting viral production and the other control input represents the efficiency of drug therapy in blocking new infection. The proposed controller ensures the stability and robust performance in the presence of parametric and non‐parametric uncertainties (and/or bounded disturbances). The global stability and tracking convergence of the process are investigated by employing the Lyapunov theorem. The performance of the proposed controller is evaluated using simulations by considering different levels of uncertainties. Based on the obtained results, the proposed strategy can achieve its desired objectives with different cases of uncertainties. [ABSTRACT FROM AUTHOR]

Abstract: In aquatic habitats, viral lysis of prokaryotic cells lowers the overall efficiency of the microbial loop, by which dissolved organic carbon is transfered to higher trophic levels. Mixing of water masses in the dark ocean occurs on a global scale and may have far reaching consequences for the different prokaryotic and virus communities found in these waters by altering the environmental conditions these communities experience. We hypothesize that mixing of deep ocean water masses enhances the lytic activity of viruses infecting prokaryotes. To address this hypothesis, major deep‐sea water masses of the Atlantic Ocean such as North Atlantic Deep Water, Mediterranean Sea Overflow Water, Antarctic Intermediate Water, and Antarctic Bottom Water were sampled at five locations. Prokaryotic cells from these samples were collected by filtration and subsequently incubated in virus‐reduced water from either the same (control) or a different water mass (transplantation treatment). Additionally, mixtures of prokaryotes obtained from two different water masses were incubated in a mixture of virus‐reduced water from the same water masses (control) or in virus‐reduced water from the source water masses separately (mixing treatments). Pronounced differences in productivity‐related parameters (prokaryotic leucine incorporation, prokaryotic and viral abundance) between water masses caused strong changes in viral lysis of prokaryotes. Often, mixing of water masses increased viral lysis of prokaryotes, indicating that lysogenic viruses were induced into the lytic cycle. Mixing‐induced changes in viral lysis had a strong effect on the community composition of prokaryotes and viruses. Our data show that mixing of deep‐sea water masses alters levels of viral lysis of prokaryotes and in many cases weakens the efficiency of the microbial loop by enhancing the recycling of organic carbon in the deep ocean. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV.

Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3H-thymidine incubations for the determination of viral production rates, and the use of 3H-thymidine versus 3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 107 viruses g-1 h-1, and were similar but overall less variable compared to those obtained by the 3H-thymidine method (0.3 to 9.0 × 107 viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3-35.1 and 9.3-34.6 ngC g-1h-1 using the 3H-thymidine or 3H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55-81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers, also reducing time to results and overcoming issues related to radioactive waste generation.