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Human exploration of the solar system will expose crew members to galactic cosmic radiation (GCR), with a potential for adverse health effects. GCR particles (protons and ions) move at nearly the speed of light and easily penetrate space station walls, as well as the human body. Previously, we have shown reactivation of latent herpesviruses, including herpes simplex virus, Varicella zoster virus, Epstein-Barr virus, and cytomegalovirus (CMV), during stays at the International Space Station. Given the prevalence of latent CMV and the known propensity of space radiation to cause alterations in many cellular processes, we undertook this study to understand the role of GCR in reactivating latent CMV. Latently infected Kasumi cells with CMV were irradiated with 137 Cs gamma rays, 150 MeV protons, 600 MeV/n carbon ions, 600 MeV/n iron ions, proton ions, and simulated GCR. The CMV copy number increased significantly in the cells exposed to radiation as compared with the non-irradiated controls. Viral genome sequencing did not reveal significant nucleotide differences among the compared groups. However, transcriptome analysis showed the upregulation of transcription of the UL49 ORF, implicating it in the switch from latent to lytic replication. These findings support our hypothesis that GCR may be a strong contributor to the reactivation of CMV infection seen in ISS crew members.

The therapeutic efficacy of oncolytic adenoviruses (OAs) relies on efficient viral transduction and replication. However, the limited expression of coxsackie-adenovirus receptors in many tumors, along with the intracellular antiviral signaling, poses significant obstacles to OA infection and oncolysis. Here, we present sonosensitizer-armed OAs (saOAs) that potentiate the antitumor efficacy of oncolytic virotherapy through sonodynamic therapy-augmented virus replication. The saOAs could not only efficiently infect tumor cells via transferrin receptor-mediated endocytosis but also exhibit enhanced viral replication and tumor oncolysis under ultrasound irradiation. We revealed that the sonosensitizer loaded on the viruses induced the generation of ROS within tumor cells, which triggered JNK-mediated autophagy, ultimately leading to the enhanced viral replication. In mouse models of malignant melanoma, the combination of saOAs and sonodynamic therapy elicited a robust antitumor immune response, resulting in significant inhibition of melanoma growth and improved host survival. This work highlights the potential of sonodynamic therapy in enhancing the effectiveness of OAs and provides a promising platform for fully exploiting the antitumor efficacy of oncolytic virotherapy.

This experimental study aimed to evaluate the antiviral and synergistic effects of photoenergy irradiation on human herpes simplex virus type I (HSV-1) infection. We assessed viral replication, plaque formation, and relevant viral gene expression to examine the antiviral and synergistic effects of blue light (BL) with acyclovir treatment. Our results showed that daily BL (10 J/cm 2 ) irradiation inhibited plaque-forming ability and decreased viral copy numbers in HSV-1-infected monkey kidney epithelial Vero cells and primary human oral keratinocyte (HOK) cells. Combined treatment with the antiviral agent acyclovir and BL irradiation increased anti-viral activity, reducing viral titers and copy numbers. In particular, accumulated BL irradiation suppressed characteristic viral genes including UL19 and US6, and viral DNA replication-essential genes including UL9, UL30, UL42, and UL52 in HOK cells. Our results suggest that BL irradiation has anti-viral and synergistic properties, making it a promising therapeutic candidate for suppressing viral infections in clinical trials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19)., (© 2021. The Author(s).)

Methylene blue is an FDA (Food and Drug Administration) and EMA (European Medicines Agency) approved drug with an excellent safety profile. It displays broad-spectrum virucidal activity in the presence of UV light and has been shown to be effective in inactivating various viruses in blood products prior to transfusions. In addition, its use has been validated for methemoglobinemia and malaria treatment. In this study, we first evaluated the virucidal activity of methylene blue against influenza virus H1N1 upon different incubation times and in the presence or absence of light activation, and then against SARS-CoV-2. We further assessed the therapeutic activity of methylene blue by administering it to cells previously infected with SARS-CoV-2. Finally, we examined the effect of co-administration of the drug together with immune serum. Our findings reveal that methylene blue displays virucidal preventive or therapeutic activity against influenza virus H1N1 and SARS-CoV-2 at low micromolar concentrations and in the absence of UV-activation. We also confirm that MB antiviral activity is based on several mechanisms of action as the extent of genomic RNA degradation is higher in presence of light and after long exposure. Our work supports the interest of testing methylene blue in clinical studies to confirm a preventive and/or therapeutic efficacy against both influenza virus H1N1 and SARS-CoV-2 infections., (© 2021. The Author(s).)

The RNA-sensing pathway contributes to type I interferon (IFN) production induced by DNA damaging agents. However, the potential involvement of RNA sensors in DNA repair is unknown. Here, we found that retinoic acid-inducible gene I (RIG-I), a key cytosolic RNA sensor that recognizes RNA virus and initiates the MAVS-IRF3-type I IFN signaling cascade, is recruited to double-stranded breaks (DSBs) and suppresses non-homologous end joining (NHEJ). Mechanistically, RIG-I interacts with XRCC4, and the RIG-I/XRCC4 interaction impedes the formation of XRCC4/LIG4/XLF complex at DSBs. High expression of RIG-I compromises DNA repair and sensitizes cancer cells to irradiation treatment. In contrast, depletion of RIG-I renders cells resistant to irradiation in vitro and in vivo. In addition, this mechanism suggests a protective role of RIG-I in hindering retrovirus integration into the host genome by suppressing the NHEJ pathway. Reciprocally, XRCC4, while suppressed for its DNA repair function, has a critical role in RIG-I immune signaling through RIG-I interaction. XRCC4 promotes RIG-I signaling by enhancing oligomerization and ubiquitination of RIG-I, thereby suppressing RNA virus replication in host cells. In vivo, silencing XRCC4 in mouse lung promotes influenza virus replication in mice and these mice display faster body weight loss, poorer survival, and a greater degree of lung injury caused by influenza virus infection. This reciprocal regulation of RIG-I and XRCC4 reveals a new function of RIG-I in suppressing DNA repair and virus integration into the host genome, and meanwhile endues XRCC4 with a crucial role in potentiating innate immune response, thereby helping host to prevail in the battle against virus.

The potential virucidal effects of UV-C irradiation on SARS-CoV-2 were experimentally evaluated for different illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density comparable to that observed in SARS-CoV-2 infection, an UV-C dose of just 3.7 mJ/cm 2 was sufficient to achieve a more than 3-log inactivation without any sign of viral replication. Moreover, a complete inactivation at all viral concentrations was observed with 16.9 mJ/cm 2 . These results could explain the epidemiological trends of COVID-19 and are important for the development of novel sterilizing methods to contain SARS-CoV-2 infection.

The coronavirus disease-2019 (COVID-19) pandemic caused by SARS-CoV-2 has exacted an enormous toll on healthcare systems worldwide. The cytokine storm that follows pulmonary infection is causally linked to respiratory compromise and mortality in the majority of patients. The sparsity of viable treatment options for this viral infection and the sequelae of pulmonary complications have fueled the quest for new therapeutic considerations. One such option, the long-forgotten idea of using low-dose radiation therapy, has recently found renewed interest in many academic centers. We outline the scientific and logistical rationale for consideration of this option and the mechanistic underpinnings of any potential therapeutic value, particularly as viewed from an immunological perspective. We also discuss the preliminary and/or published results of prospective trials examining low-dose radiation therapy for COVID-19., (© The Author(s) 2020. Published by Oxford University Press.)

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980's, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 μmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 μmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur., Competing Interests: The authors have declared that no competing interests exist.

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock, especially poultry and pigs. This study aimed to investigate how to inactivate IAVs by using different ultraviolet-light-emitting diodes (UV-LEDs). We developed sterilization equipment with light-emitting diodes (LEDs) those peak wavelengths were 365 nm (UVA-LED), 310 nm (UVB-LED), and 280 nm (UVC-LED). These UV-LED irradiations decreased dose fluence-dependent plaque-forming units of IAV H1N1 subtype (A/Puerto Rico/8/1934) infected Madin-Darby canine kidney (MDCK) cells, but the inactivation efficiency of UVA-LED was significantly lower than UVB- and UVC-LED. UV-LED irradiations did not alter hemagglutination titer, but decreased accumulation of intracellular total viral RNA in infected MDCK cells was observed. Additionally, UV-LED irradiations suppressed the accumulation of intracellular mRNA (messenger RNA), vRNA (viral RNA), and cRNA (complementary RNA), as measured by strand-specific RT-PCR. These results suggest that UV-LEDs inhibit host cell replication and transcription of viral RNA. Both UVB- and UVC-LED irradiation decreased focus-forming unit (FFU) of H5N1 subtype (A/Crow/Kyoto/53/2004), a highly pathogenic avian IAV (HPAI), in infected MDCK cells, and the amount of FFU were lower than the H1N1 subtype. From these results, it appears that IAVs may have different sensitivity among the subtypes, and UVB- and UVC-LED may be suitable for HPAI virus inactivation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.

More than 70 human adenovirus types were identified divided into 7 different species (A-G). Diseases caused by human adenoviruses are type-dependent and can range from mild to severe respiratory infections, gastrointestinal infections or eye infections such as epidemic keratoconjunctivitis. Unfortunately there is no specific anti-adenovirus therapy available. Here we addressed the question whether treatment with cold atmospheric plasma (CAP) for anti-adenoviral therapy such as virus-mediated ulcerations may be feasible. CAP has already been explored for the treatment of dermatological diseases such as chronic wounds. To investigate whether CAP is an effective antiviral tool, purified human adenovirus types derived from different human adenovirus species (HAdV -4, -5, -20, -35, -37, -50) tagged with luciferase were treated with defined dosages of plasma. The CAP treatment was varied by incrementally increasing the time span of CAP treatment. After CAP treatment, the virus containing solution was added to eukaryotic cells and the viral load was determined by measurement of luciferase expression levels. Through the plasma treatment the adenovirus driven luciferase expression directly correlating with adenovirus transduction efficiencies could be reduced for HAdV-5 and HAdV-37. Plasma treatment had no influence on adenovirus derived luciferase expression levels for HAdV-4 and HAdV-50 and it even had a positive effect on luciferase expression levels for HAdV-20 and HAdV-35. These results suggest that CAP has a type dependent effect on adenoviruses and that infectivity can be even increased for certain adenovirus types. Further studies should address the mechanisms behind this phenomenon. In summary we demonstrate that CAP may represent an interesting option for antiviral treatment in a virus type dependent manner., Competing Interests: The authors have declared that no competing interests exist.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We investigated pH optimum of fusion, temperatures of HA heat inactivation, in vivo and in vitro replication kinetics, and connectivity with panel of sera of survivors patients in different epidemic seasons of the previously obtained influenza H1 escape mutants. Our results showed that N133D (H3 numbering) mutation significantly lowered the pH of fusion optimum. Several amino acid substitutions, including K163 N, Q192 L, D190E, G228E, and K285 M, reduced the stability of HA as determined by heat inactivation, whereas A198E substitution is associated with significant increase in HA thermostability compared to the wild-type virus. We found that amino acid change D190 N was associated with a significant decrease in viral growth in eggs and mice. Our potential antigenic variants, except readapted variant, which contained A198E mutation, did not reach fixation in infected people. Overall, a co-variation between antigenic specificity and different HA phenotypic properties was demonstrated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Middle East respiratory syndrome-coronavirus (MERS-CoV) is a novel zoonotic pathogen. Although the potential for MERS-CoV transmission through blood transfusion is not clear, MERS-CoV was recognized as a pathogen of concern for the safety of the blood supply especially after its detection in whole blood, serum, and plasma of infected individuals. Here we investigated the efficacy of amotosalen and ultraviolet A light (UVA) to inactivate MERS-CoV in fresh-frozen plasma (FFP)., Study Design and Methods: Pooled FFP units were spiked with a recent clinical MERS-CoV isolate. Infectious and genomic viral titers were determined in plasma before and after inactivation with amotosalen/UVA treatment by plaque assay and reverse transcription-quantitative polymerase chain reaction, respectively. In addition, residual replicating or live virus after inactivation was examined by passaging in the permissive Vero E6 cells., Results: The mean MERS-CoV infectious titer in pretreatment samples was 4.67 ± 0.25 log plaque-forming units (pfu)/mL, which was reduced to undetectable levels after inactivation with amotosalen/UVA demonstrating a mean log reduction of more than 4.67 ± 0.25 pfu/mL. Furthermore, inoculation of inactivated plasma on Vero E6 cells did not result in any cytopathic effect (CPE) even after 7 days of incubation and three consecutive passages, nor the detection of MERS RNA compared to pretreatment samples which showed complete CPE within 2 to 3 days postinoculation and log viral RNA titer ranging from 9.48 to 10.22 copies/mL in all three passages., Conclusion: Our data show that amotosalen/UVA treatment is a potent and effective way to inactivate MERS-CoV infectious particles in FFP to undetectable levels and to minimize the risk of any possible transfusion-related MERS-CoV transmission., (© 2017 AABB.)

Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter Micromonas . We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 ± 2 vs. 191 ± 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term (seasonal cycles) and long term (global warming)., Competing Interests: The authors declare no conflict of interest.

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425-428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development., Importance: Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional analysis to demonstrate that viral prM-E genes are the main determinants for the high stability of ZIKV. The extra hydrogen-bond interaction (observed in the high-resolution structure) between ZIKV E proteins did not enhance virion stability, whereas the extended loop of E protein (CD loop in domain III) was essential for ZIKV assembly. More importantly, we found that a chimeric ZIKV with DENV-2 prM-E genes and a chimeric DENV-2 with ZIKV prM-E genes were highly attenuated in A129 mice. Mice immunized with these chimeric viruses generated robust neutralizing antibody responses and were fully protected from DENV-2 and ZIKV challenge, respectively, indicating that these chimeric viruses could be further developed as vaccine candidates., (Copyright © 2017 Xie et al.)

Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET) proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP), thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2) and its host (Prochlorococcus MED4) throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase)-implicated in protecting the photosynthetic membrane from light stress-were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin-NADP+ reductase (FNR), which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under infection, with ATP from PET and NADPH from the PPP producing the energy and reducing equivalents for phage nucleotide biosynthesis and replication., Competing Interests: The authors have declared that no competing interests exist.

The Arenaviridae is a diverse and growing family of viruses that already includes more than 25 distinct species. While some of these viruses have a significant impact on public health, others appear to be non-pathogenic. At present little is known about the host cell responses to infection with different arenaviruses, particularly those found in the New World; however, apoptosis is known to play an important role in controlling infection of many viruses. Here we show that infection with Tacaribe virus (TCRV), which is widely considered the prototype for non-pathogenic arenaviruses, leads to stronger induction of apoptosis than does infection with its human-pathogenic relative Junín virus. TCRV-induced apoptosis occurred in several cell types during late stages of infection and was shown to be caspase-dependent, involving the activation of caspases 3, 7, 8 and 9. Further, UV-inactivated TCRV did not induce apoptosis, indicating that the activation of this process is dependent on active viral replication/transcription. Interestingly, when apoptosis was inhibited, growth of TCRV was not enhanced, indicating that apoptosis does not have a direct negative effect on TCRV infection in vitro. Taken together, our data identify and characterize an important virus-host cell interaction of the prototypic, non-pathogenic arenavirus TCRV, which provides important insight into the growing field of arenavirus research aimed at better understanding the diversity in responses to different arenavirus infections and their functional consequences.

Viruses are ubiquitous drivers of microbial ecology and evolution and contribute to biogeochemical cycling. Attention to these attributes has been more substantial for marine viruses than viruses of other environments. Microscopy-based investigation of the viral communities from two cold, hypersaline Arctic springs was undertaken to explore the effects of these conditions on microbe-viral ecology. Sediments and water samples were collected along transects from each spring, from anoxic spring outlets through oxygenated downstream channels. Viral abundance, virus-microbe ratios and modelled virus-microbe contact rates were lower than comparable aqueous and sedimentary environments and most similar to deep subsurface sediments. No individual cell from either spring was visibly infected. Viruses in these springs appear to play a smaller role in controlling microbial populations through lytic activity than in marine water column or surface sedimentary environments. Relief from viral predation indicates the microbial communities are primarily controlled by nutrient limitation. The similarity of these springs to deep subsurface sediments suggests a biogeographic divide in viral replication strategy in marine sediments., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The use of highly active antiretroviral therapy against HIV-1 for last two decades has reduced mortality of patients through extension of nonsymptomatic phase of infection. However, HIV-1 can be preserved in long-lived resting CD4(+) T cells, which form a viral reservoir in infected individuals, and potentially in macrophages and astrocytes. Reactivation of viral replication is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus (shock and kill strategy). In this opinion piece, we consider potential application of therapeutic doses of irradiation, the well-known and effective stress signal that induces DNA damage and activates cellular stress response, to resolve two problems: activate HIV-1 replication and virion production in persistent reservoirs under cART and deplete infected cells through selective cell killing using DNA damage responses.

Unlabelled: Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments., Importance: Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Escherichia coli O157:H7 causes serious foodborne infections warranting the development of effective control measures. One control option is to use bacteriophages (phages), which are regarded as safe to humans and an environmentally friendly alternative to chemical antimicrobials. One of the few remaining safety concerns is the potential for phages to facilitate genetic exchange between bacteria so resulting in undesirable mobilisation of genes. UV treatment of phages causes a rapid loss in their ability to replicate, while maintaining their antibacterial activity, and so the use of UV-treated phages could be an alternative to the use of viable phages. Data presented here show the inactivation of E. coli O157:H7 by UV-treated phages in milk and on the surface of raw and cooked meat. A minimum concentration of approximately 10(5) PFU cm(-2) (pre-UV treatment titre) of UV-treated phages was required before inactivation of E. coli O157:H7 on the surface of meat was measurable, and 1-2 log10 CFU cm(-2) reductions were typically obtained at concentrations of around 10(7) UV-treated phages cm(-2) (pre-UV treatment titre). Inactivation of E. coli O157:H7 by UV-treated phages was less than that for untreated phages. The production of UV-treated phages was not optimised and it is possible that better reductions in pathogen concentration could be achieved for the same input UV-treated phages concentrations., (© The Author(s) 2014.)

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines., Importance: By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Vaccination involves inoculation of a subject with a disabled disease-causing microbe or parts thereof. While vaccination has been highly successful, we still lack sufficiently effective vaccines for important infectious diseases. We propose that a more complete immune response than that elicited from a vaccine may be obtained from immunization with a disease-causing virus modified to subject replication-essential genes to the control of a gene switch activated by non-lethal heat in the presence of a drug-like compound. Upon inoculation, strictly localized replication of the virus would be triggered by a heat dose administered to the inoculation site. Activated virus would transiently replicate with an efficiency approaching that of the disease-causing virus and express all viral antigens. It may also vector heterologous antigens or control co-infecting microbes.

Background: Arboviruses are an emerging threat to transfusion safety and rates of infection are likely to increase with the increased rainfall associated with climate change. Arboviral infections are common in Australia, where Ross River virus (RRV), Barmah Forest virus (BFV), and Murray Valley encephalitis virus (MVEV), among others, have the potential to cause disease in humans. The use of pathogen reduction technology (PRT) may be an alternative approach for blood services to manage the risk of arboviral transfusion transmission. In this study, the effectiveness of the Mirasol PRT (Terumo BCT) system at inactivating RRV, BFV, and MVEV in buffy coat (BC)-derived platelets (PLTs) was investigated., Study Design and Methods: BC-derived PLT concentrates in additive solution (SSP+) were spiked with RRV, BFV, or MVEV and then treated with the Mirasol PRT system. The level of infectious virus was determined before and after treatment, and the reduction in viral infectivity was calculated., Results: Treatment with PRT (Mirasol) reduced the amount of infectious virus of all three arboviruses. The greatest level of inactivation was observed for RRV (2.33 log; 99.25%), followed by BFV (1.97 log; 98.68%) and then MVEV (1.83 log; 98.42%)., Conclusion: Our study demonstrates that treatment of PLT concentrates with PRT (Mirasol) reduces the infectious levels of RRV, BFV, and MVEV. The relevance of the level of reduction required to prevent disease transmission by transfusion has not been fully defined and requires further investigation. In the face of a changing climate, with its associated threat to blood safety, PRT represents a proactive approach for maintaining blood safety., (© 2014 AABB.)

Unlabelled: Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following the identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the classical Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts--if any exist besides boid snakes themselves--are not yet known. In this report, we use University of Helsinki virus (UHV; a virus that we isolated from a Boa constrictor with BIBD) to show that BIBDAV can also replicate effectively in mammalian cells, including human cells, provided they are cultured at 30°C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transferring infected cells from 30°C to 37°C ambient temperature resulted in progressive declines in IB formation and in the amounts of viral NP and RNA, suggesting that BIBDAV growth is limited at 37°C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30°C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature, such as snakes., Importance: The newly discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in captive boid snakes, but their possible reservoir host(s) have not yet been identified. Here we show, using University of Helsinki virus as a model, that these viruses are able to infect mammalian (including human) and arthropod cells. Our results provide in vitro proof of the considerable ability of arenaviruses to cross species barriers. However, our data indicate that BIBDAV growth occurs at 30°C but is inhibited at 37°C, implying that crossing of the species barrier would be hindered by the body temperature of mammalian species., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Viral hemorrhagic septicemia virus (VHSV) has four genotypes (I-IV) and sub-lineages within genotype I and IV. Using a reverse genetics approach, we explored the importance of the L gene for growth characteristics at different temperatures following interchange of the L gene within genotype IV (IVa and IVb) strains. VHSV strains harboring heterologous L gene were recovered and we show that the L gene determines growth characteristics at different temperatures in permissive cell lines., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults., Importance: There is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccine (pLAIV) viruses and observed that the pLAIV viruses consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIV viruses may account for their restricted replication in clinical trials., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥ 100-fold) replication at 39 °C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391 E, 581 G, and 661 T), PB2 (265 S), and NP (34 G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04(ts sig) and CA07(ts sig) viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39 °C, but the replication of WSN(ts sig) was restricted ≥ 100-fold compared to that at 33 °C. Reassortant CA07(ts sig) viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥ 100-fold at 39 °C. In ferrets, the CA07(ts sig) virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07(ts sig) virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses., Importance: Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Koi herpesvirus disease (KHVD) is an emerging and highly contagious viral disease of koi and common carp (Cyprinus carpio), causing mass mortalities and huge economic losses to the carp aquaculture industry. The disease has spread rapidly to 28 countries worldwide. However, mechanisms of koi herpesvirus (species Cyprinid herpesvirus 3; CyHV-3) transmission remain unclear. A potential experimental model of CyHV-3 infection in carp was used to characterise CyHV-3 in different phases of infection and to demonstrate that CyHV-3 persists in survivor fish and has the capacity to reactivate and transmit the disease to healthy fish. During acute infection, which occurred when fish were maintained at 22°C, viral genes were abundantly expressed and infectious virus was produced in association with tissue damage, clinical disease and mortality. In fish maintained at a lower temperature (11°C), viral DNA was present but viral gene expression was absent or greatly restricted, infectious virus was not recovered and there was no evidence of disease. Productive replication was re-initiated following an increase in water temperature to 22°C, resulting in 45% mortality. Shedding of reactivated virus killed 75% of cohabitating naïve fish, suggesting a potential risk for disease transmission., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Nucleotide-binding oligomerization domain 2 (NOD2) is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV) results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs) and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β) and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

We report that radiation enhances the antitumor efficacy of the oncolytic adenovirus vector VRX-007 in Syrian hamster tumors. We used tumor-specific irradiation of subcutaneous tumors and compared treatment options of radiation alone or combined with VRX-007 and cyclophosphamide (CP). Radiation therapy further augmented the VRX-007-mediated inhibition of tumor growth, in both CP-treated and non-CP-treated hamsters, even though radiation did not lead to increased viral replication in tumors when compared with those treated with VRX-007 alone. Moreover, tumor growth inhibition was similar in tumors irradiated either 1 week before or after injection with VRX-007, which suggests that radiation exerts its antitumor effect independently from vector therapy. Thus, our results demonstrate that these two therapies do not have to be provided simultaneously to enhance their combined effectiveness against subcutaneous hamster tumors.

The complete coding sequences of the four unassigned temperature-sensitive (ts) Baylor prototype rotavirus mutants (SA11ts D, H, I and J) were sequenced by deep sequencing double-stranded RNA using RNA-seq. Non-silent mutations were assigned to a specific mutant by Sanger sequencing RT-PCR products from each mutant. Mutations that led to amino acid changes were found in all genes except for genes 1 (VP1), 10 (NSP4) and 11 (NSP5/6). Based on these sequence analyses and earlier genetic analyses, the ts mutations in gene 7, which encodes the protein NSP3, were assigned to ts mutant groups I and H, and confirmed by an in vitro RNA-binding assay with recombinant proteins. In addition, ts mutations in gene 6 were assigned to tsJ. The presence of non-conservative mutations in two genes of two mutants (genes 4 and 2 in tsD and genes 3 and 7 in tsH) underscores the necessity of sequencing the whole genome of each rotavirus ts mutant prototype., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection. In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation. The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3' untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis. The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Conditionally replicating adenoviruses (CRAds) utilize tissue-specific promoters to control the expression of the early genes, E1A and E1B, to preferentially replicate and lyse tumor cells (oncolysis). Previous CRAds used in prostate cancer (PCa) gene therapy require androgens to activate prostate-specific promoters and induce viral replication. Unfortunately, these CRAds have reduced activity in patients on androgen-suppressive therapy. We describe a novel prostate-specific CRAd generated by fusing the E1A gene to the androgen receptor (AR) cDNA with a point mutation in codon 685 (C685Y). The E1A-AR fusion neutralizes the previously described mutual inhibition of E1A and AR, and the C685Y point mutation alters specificity of steroid ligand binding to the AR, such that both androgens and nonsteroidal anti-androgens can activate viral replication. We demonstrate that the mutated E1A-AR retained the ability to function in regulating AR-responsive genes and E1A-responsive viral genes. In combination therapy of virus, bicalutamide (anti-androgen) and radiation, a profound impact on cell death by viral oncolysis was seen both in vitro and tumor xenografts. To our knowledge, this is the first gene therapy engineered to be enhanced by anti-androgens and a particularly attractive adjuvant strategy for intensity-modulated radiation therapy of high-risk PCas.

Background: We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1-4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP., Methodology/principal Findings: After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors., Conclusion/significance: All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.

MEDI-559 is a recombinant live attenuated intranasal RSV vaccine candidate currently being evaluated in 5 to <24 month old RSV seronegative infants for safety and immunogenicity. MEDI-559 and the previously tested rA2cp248/404/1030ΔSH both have 5 cold-passaged mutations, 3 temperature sensitive (ts) markers designated 248, 404, and 1030, and deletion of the SH gene that collectively contribute to their attenuation and temperature sensitive growth phenotypes. However, MEDI-559 differs from rA2cp248/404/1030ΔSH by 39 silent nucleotide substitutions. Nevertheless, these viruses have comparable in vitro and in vivo phenotypes. Temperature sensitivity is monitored by the efficiency of plaque formation at elevated temperatures. The efficiency of plaque formation of MEDI-559 is reduced by ≥ 100-fold at 35 ° C and by ≥ 1000 fold at 37 °C compared to 32 °C. Passaging of MEDI-559 at temperatures up to 37 °C resulted in generation of temperature sensitive intermediate (tsi) viruses. The most frequent change was a reversion to wildtype tyrosine at the 1030 ts site followed by a less frequently observed leucine to non-wildtype serine substitution at the 248 ts site. One tsi virus had changes at both the 248 and 1030 ts sites and another tsi virus that had maintained all of the 248, 404 and 1030 ts sites had two novel changes (Asp158Gly and Ser1313Cys) in the polymerase (L) gene. Asp158Gly and Ser1313Cys singly or in combination in the MEDI-559 genetic background were confirmed to result in a tsi growth phenotype. All the tsi viruses have small plaque phenotypes and are highly attenuated in the lungs of cotton rats., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Combinatorial therapy is the current trend of the development of novel cancer treatments due to the high heterogenous nature of solid tumors. In this study, we investigated the effects of the combined use of a conditionally replicating adenovirus carrying IL-24 (ZD55-IL-24) and radiotherapy on the proliferation and apoptosis of melanoma A375 cells in vitro and in vivo. Compared with either agent used alone, ZD55-IL-24 combined with radiotherapy significantly inhibited cell proliferation, accompanied with increased apoptosis. Radiotherapy did not affect the expression of IL-24 and E1A of ZD55-IL-24-treated cells, but increased the expression of Bax, promoted the activation of caspase-3, while decreasing Bcl-2 levels. Thus, this synergistic effect of ZD55-IL-24 in combination with radiotherapy provides a novel strategy for the development of melanoma therapies, and is a promising approach for further clinical development., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40°C, failed to replicate to detectable levels in mice. A total of 18 non-synonymous mutations have been identified in tsm5. In a previous study, a mutation (C890Y) identified in the M70 primase gene, when introduced into the wt M70 primase, resulted in a mutant with reduced viral replication at 40°C in vitro and which was severely attenuated in vivo. Five other previously identified mutations may also contribute to the tsm5 phenotype: (1) an A658S mutation in a protein expressed by the M27 ORF; (2) a V54I mutation in M36; (3) a Y565* mutation in m139; (4) a V195M mutation in m141; and (5) an M232I mutation in m143. In the present study, the above-mentioned mutations were introduced individually (M27, M36, m139, m141, m143) or together (M27/M36) into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. Growth in culture revealed that, apart from the double mutant (M27 and M36) and the m139 mutant, the introduced mutations in the above-mentioned genes did not show a temperature-sensitive phenotype in MEF or Raw 264.7 macrophage cells compared to their revertants or the wt virus. In contrast, replication of the M27/M36 double mutant was drastically reduced in MEFs at 40°C and in macrophages at 37°C. Replication of the m139 mutant was reduced in MEF cells at 40°C but not in macrophages. Thus, at least three further mutations contribute to the tsm5 phenotype., (Copyright © 2012 Wiley Periodicals, Inc.)

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30°C and 33°C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39°C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist(®)-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression., Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured., Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression., Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oncolytic viruses have been combined with standard cancer therapies to increase therapeutic efficacy. Given the sequential activation of herpes viral genes (herpes simplex virus-1, HSV-1) and the temporal cellular changes induced by ionizing radiation, we hypothesized an optimal temporal sequence existed in combining oncolytic HSV-1 with ionizing radiation. Murine U-87 glioma xenografts were injected with luciferase encoding HSV-1, and ionizing radiation (IR) was given at times before or after viral injection. HSV-1 replication and tumor-volume response were followed. Radiation given 6-9 h after HSV-1 injection resulted in maximal viral luciferase expression and infectious viral production in tumor xenografts. The greatest xenograft regression was also seen with radiation given 6 h after viral injection. We then tested if HSV-1 replication had a dose response to ionizing radiation. HSV-1 luciferase expression exhibited a dose response as xenografts were irradiated from 0 to 5 Gy. There was no difference in viral luciferase expression as IR dose increased from 5 Gy up to 20 Gy. These results suggest that the interaction of IR with the HSV-1 lytic cycle can be manipulated for therapeutic gain by delivering IR at a specific time within viral replicative cycle.

The presence of West Nile virus (WNV) was first documented in California, USA, during the summer of 2003, and subsequently the virus has become endemic throughout the state. Sequence analysis has demonstrated that the circulating strains are representative of the North American (WN02) genotype that has displaced the East Coast genotype (NY99). A recent study has indicated that enhanced vector competence at elevated temperatures may have played a role in the displacement of the East Coast genotype by WN02. In the current study, four WN02 strains from California, including an initial 2003 isolate (COAV997), were compared to strain NY99 in growth curve assays in mosquito and duck embryonic fibroblast (DEF) cell lines at differing, biologically relevant temperatures to assess the relative temperature sensitivities of these natural isolates. COAV997 was significantly debilitated in viral replication in DEF cells at 44 °C. Full-length sequence comparison of COAV997 against the NY99 reference strain revealed non-synonymous mutations in the envelope glycoprotein (V159A), non-structural protein 1 (NS1) (K110N) and non-structural protein 4A (NS4A) (F92L), as well as two mutations in the 3' UTR: C→T at nt 10 772 and A→G at nt 10 851. These non-synonymous mutations were introduced into the NY99 viral backbone by site-directed mutagenesis. A mutant containing the NS1-K110N and NS4A-F92L mutations exhibited a debilitated growth phenotype in DEF cells at 44 °C, similar to that of COAV997. One explanation for the subsistence of this genotype is that COAV997 was obtained from an area of California where avian host species might not present elevated temperatures. These data indicate that the NS1 and NS4A mutations identified in some WN02 isolates could reduce thermal stability and impede replication of virus at temperatures observed in febrile avian hosts.

Background: Ultrasound has been shown to increase the efficiency of gene expression from retroviruses, adenoviruses and adeno-associated viruses. The effect of ultrasound to stimulate cell membrane permeabilization on infection with an oncolytic herpes simplex virus type 1 (HSV-1) was examined., Results: Vero monkey kidney cells were infected with HSV-1 and exposed to 1 MHz ultrasound after an adsorption period. The number of plaques was significantly greater than that of the untreated control. A combination of ultrasound and microbubbles further increased the plaque number. Similar results were obtained using a different type of HSV-1 and oral squamous cell carcinoma (SCC) cells. The appropriate intensity, duty cycle and time of ultrasound to increase the plaque number were 0.5 W/cm², 20% duty cycle and 10 sec, respectively. Ultrasound with microbubbles at an intensity of 2.0 W/cm², at 50% duty cycle, or for 40 sec reduced cell viability., Conclusion: These results indicate that ultrasound promotes the entry of oncolytic HSV-1 into cells. It may be useful to enhance the efficiency of HSV-1 infection in oncolytic virotherapy.

Each segment of the influenza A virus (IAV) genome contains conserved sequences at the 5'- and 3'-terminal ends, which form the promoter region necessary for polymerase binding and initiation of RNA synthesis. Although several models of interaction have been proposed it remains unclear if these two short, partially complementary, and highly conserved sequences can form a stable RNA duplex at physiological temperatures. First, our time-resolved FRET analysis revealed that a 14-mer 3'-RNA and a 15-mer 5'-RNA associate in solution, even at 42 °C. We also found that a nonfunctional RNA promoter containing the 3'-G3U mutation, as well as a promoter containing the compensatory 3'-G3U/C8A mutations, was able to form a duplex as efficiently as wild type. Second, UV melting analysis demonstrated that the wild-type and mutant RNA duplexes have similar stabilities in solution. We also observed an increase in thermostability for a looped promoter structure. The absence of differences in the stability and binding kinetics between wild type and a nonfunctional sequence suggests that the IAV promoter can be functionally inactivated without losing the capability to form a stable RNA duplex. Finally, using uridine specific chemical probing combined with mass spectrometry, we confirmed that the 5' and 3' sequences form a duplex which protects both RNAs from chemical modification, consistent with the previously published panhandle structure. These data support that these short, conserved promoter sequences form a stable complex at physiological temperatures, and this complex likely is important for polymerase recognition and viral replication.

Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.