Temperature-sensitive Virus Research Articles

Rejuvenation of human mesenchymal stem cells using a nonintegrative and conditionally removable Sendai virus vector

Human mesenchymal stem cells (hMSCs) with extended lifespan and differentiation potential that can recapitulate in vivo characteristics could significantly contribute to basic research, drug development, and cell therapy. Specifically, they could ensure a stable supply of specific cellular resources, and possibly extracellular vesicles. Here, we established a technology for extending the lifespan while maintaining differentiation potential, termed “rejuvenation,” of hMSCs (rej-hMSCs) using nonintegrative and conditionally removable temperature-sensitive Sendai virus (SeV) vectors. Various immortalizing factors (i.e., Bmi-1, hTERT, SV40T, and/or HPV E6/E7) were first introduced by the SeV vector into the cells. A combination of three SeVs with Bmi-1, hTERT, or SV40T conferred markedly improved cell proliferation and cloning ability while maintaining differentiation potential and a normal karyotype. An extended lifespan was also demonstrated in other cell types. The rejuvenation of long-passaged or aged hMSCs was also confirmed. SeV vectors were rapidly removed as a function of cell doubling by increasing the temperature from 35 °C to 37 °C or higher, while proliferative ability was maintained. Following FACS sorting, the complete removal of SeV vectors was confirmed by qPCR analyses. Therefore, our cell rejuvenation technology could contribute to research and clinical applications by enabling the supply of modified cells without damaging host chromosomes.

Successful Ex Vivo Telomere Elongation with Exg-001 in a Patient with a Dyskeratosis Congenita

We report a first-in-human gene therapy strategy to extend telomere length in patients with telomere biology disorders. Patients with telomere biology disorders, including dyskeratosis congenita, have extremely short telomeres leading to premature cellular senescence and increased risk of bone marrow failure. Zinc Finger and SCAN Domain Containing 4 (ZSCAN4) is a protein that regulates telomere elongation, enhances genome stability, and is expressed transiently in preimplantation embryos, germ cells during meiosis, embryonic stem cells, and rare adult tissue stem cells. EXG-001 is a non-integrating temperature-sensitive non-transmissible Sendai virus vector encoding for human ZSCAN4. We demonstrated that the transient exposure to ZSCAN4 via EXG-001 extended telomeres of primary fibroblast cells derived from dyskeratosis congenita patients as well as healthy human CD34+ cells. However, we did not know whether EXG-001 can extend short telomeres of CD34+ cells of dyskeratosis congenita patients. We translated this work in a first-in-human clinical trial (NCT04211714), in which autologous CD34+ hematopoietic stem cells (HSC) were collected by pheresis from an adult subject with dyskeratosis congenita over two days after mobilization with plerixafor and filgrastim with IRB approval and consent. 2.8x106 CD34/kg were collected and treated with EXG-001 ex vivo in a functionally closed tubing system using the CliniMACS Prodigy (Miltenyi Biotec). EXG-001 treatment increased telomere length of the CD34+ cells of study subject by 1.24-fold, bringing them into the healthy normal control range of CD34+ cell telomere length (Figure 1A). To address the concern of effects on terminal colony forming unit studies were performed and demonstrated maintenance of normal differentiation potential after treatment (Figure 1B; EXG34217). 0.85x106 CD34/kg autologous treated HSC (EXG34217) were reinfused into the subject in February 2022 without conditioning regimen and further in vivo studies are in progress. This novel approach is genotype and mutation independent and uses modified autologous stem cells in the absence of conditioning regimen or immunosuppression which is highly desirable in this radiation and chemotherapy sensitive population. These data support further investigation and translation of telomere elongation via ZSCAN4 exposure by EXG-001 in patients with telomere biology disorders as a potential therapeutic intervention for the prevention or treatment of bone marrow failure. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correlation Study of the Most Important Environmental Influencing Factors on the Razi MMR Vaccine.

Measles, mumps, and rubella (MMR) are among the most important viral infectious diseases in Iran and neighboring countries. After using a trivalent vaccine for these three diseases for a long time, in recent years, these diseases have been significantly controlled in Iran. One of the important points of storing the vaccine is that the vaccine strains are highly temperature-sensitive viruses. Due to tropical climatic conditions in Iran, the cold chain may not be achievable during the storage and transmission of the MMR vaccine. Therefore, the efficacy of the vaccine may be affected. This study aimed to evaluate the MMR vaccine potency at different temperatures (stress tests) and frequent light exposures. All quality control tests in the form of stability studies were performed on the samples from three consecutive batches produced during a full-scale Razi production. The samples were stored at 2-8, 22-25, 35-37, and 42-45°C in specific time intervals, exposed to frequent light, and underwent freezing/thawing conditions. According to the results, the storage of the vaccine at high temperatures caused a decrease in potency and increased moisture content in the vaccine vials. The best temperature for maintenance and transportation of MMR is 2-8°C. The time and frequency of light exposure may affect the vaccine potency. Based on the sensitivity of the vaccine strains to environmental conditions, the development of plans for storage and transportation of vaccines in different situations and training the vaccine injection staff seem necessary.

Constitutive activation of S1P receptors at the trans-Golgi network is required for surface transport carrier formation

SummaryThe importance of the G-protein βγ subunits in the regulation of cargo transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and β-arrestin association before internalization. Continuously activated S1P receptors in a manner dependent on S1P at the TGN kept transmitting G-protein signals including the βγ subunits supply necessary for transport carrier formation at the TGN destined for the PM.

Simple derivation of skeletal muscle from human pluripotent stem cells using temperature-sensitive Sendai virus vector.

Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature‐sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV‐Myod1), a myogenic master transcription factor. SeV‐Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre‐like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell‐targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human‐induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.

Zika virus outbreak in Brazil under current and future climate

IntroductionZika virus (ZIKV) is primarily transmitted byAedes aegypti and Aedes albopictus mosquitoes between humans and non-human primates. Climate change may enhance virus reproduction in Aedes spp. mosquito populations, resulting in intensified ZIKV outbreaks. The study objective was to explore how an outbreak similar to the 2016 ZIKV outbreak in Brazil might unfold with projected climate change. MethodsA compartmental infectious disease model that included compartments for humans and mosquitoes was developed to fit the 2016 ZIKV outbreak data from Brazil using least squares optimization. To explore the impact of climate change, published polynomial relationships between temperature and temperature-sensitive mosquito population and virus transmission parameters (mosquito mortality, development rate, and ZIKV extrinsic incubation period) were used. Projections for future outbreaks were obtained by simulating transmission with effects of projected average monthly temperatures on temperature-sensitive model parameters at each of three future time periods: 2011–2040, 2041–2070, and 2071–2100. The projected future climate was obtained from an ensemble of regional climate models (RCMs) obtained from the Co-Ordinated Regional Downscaling Experiment (CORDEX) that used Representative Concentration Pathways (RCP) with two radiative forcing values, RCP4.5 and RCP8.5. A sensitivity analysis was performed to explore the impact of temperature-dependent parameters on the model outcomes. ResultsClimate change scenarios impacted the model outcomes, including the peak clinical case incidence, cumulative clinical case incidence, time to peak incidence, and the duration of the ZIKV outbreak. Comparing 2070–2100 to 2016, using RCP4.5, the peak incidence was 22,030 compared to 10,473; the time to epidemic peak was 12 compared to 9 weeks, and the outbreak duration was 52 compared to 41 weeks. Comparing 2070–2100 to 2016, using RCP8.5, the peak incidence was 21,786 compared to 10,473; the time to epidemic peak was 11 compared to 9 weeks, and the outbreak duration was 50 compared to 41weeks. The increases are due to optimal climate conditions for mosquitoes, with the mean temperature reaching 28 °C in the warmest months. Under a high emission scenario (RCP8.5), mean temperatures extend above optimal for mosquito survival in the warmest months. ConclusionOutbreaks of ZIKV in locations similar to Brazil are expected to be more intense with a warming climate. As climate change impacts are becoming increasingly apparent on human health, it is important to quantify the effect and use this knowledge to inform decisions on prevention and control strategies.

Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells

Fatal familial insomnia (FFI), genetic Creutzfeldt–Jakob disease (gCJD), and Gerstmann–Sträussler–Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

Evolution of influenza viruses based on sensitivity to temperature of replication

Introduction. The assessment of the ability of influenza viruses to replication at temperature conditions beyond optimal values approaches us to understanding the laws of their evolutionary variability. The temperature range for the reproduction of epidemic viruses is also an important indicator for choosing a rational strategy for producing attenuated reassortants for a live influenza vaccine.The purpose of the study is a retrospective analysis of the biological properties of influenza viruses from various pandemic and epidemic cycles based on their ability to replicate at temperatures beyond optimal values.Materials and methods. We studied 234 strains of human influenza A and B viruses that caused epidemics in the 20th – 21st centuries. The infectious activity of viruses at different incubation temperatures was determined in developing chicken embryos. The temperature sensitivity and cold resistance of viruses replication were estimated as a difference of infectious titers at the optimum and raised or lowered incubation temperatures.Results. Data from a retrospective study indicate that the range of sensitivity to replication temperature during the natural drift of influenza A and B pathogens is subject to regular variability that has a cyclic character.Discussion. To assess the evolution and epidemic potential of influenza viruses, it is important not only to register a change in their antigenic properties but also to take into account the temperature sensitivity of the reproduction. Both of these properties contribute to the manifestation of the virulence of the virus. Prolonged circulation of temperature-sensitive viruses can be a prerequisite for the appearance of a radically new drift variant and even shift.Conclusion. The analysis of the variability of the biological properties of influenza viruses approaches to the comprehension of possible ways of their evolution, that contributes to the development of rational methods for preventing the spread of viruses and the incidence caused by them.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

SummaryNeurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

Vaccinia Virus Phospholipase Protein F13 Promotes Rapid Entry of Extracellular Virions into Cells.

The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution.IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus.

Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus.

The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus. The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights into the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and that novel strategies to continue to improve LAIV vaccine safety should be investigated.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses.

The influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four viable mutants with single-codon deletions were generated; all of them exhibited a ts phenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serial in vitro passages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts mutants that could be used to design novel attenuated vaccines. In order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA-PB1 dimer to the nucleus, where viral replication occurs. These ts deletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

Tolerability of Nasal Delivery of Humidified and Warmed Air at Different Temperatures: A Randomised Double-Blind Pilot Study.

Objectives. Delivery of warmed, humidified air via nasal high flow therapy could potentially reduce replication of temperature-sensitive viruses in the upper respiratory tract. This study investigates whether nasal high flow therapy is well tolerated by healthy adults at 37°C and 41°C. Methods. In this randomised, double-blind, controlled crossover pilot trial, nasal high flow therapy was used to deliver humidified air at 35 L/min, at either 37°C or 41°C, for three one-hour sessions of use over one day. The alternative was delivered at least 14 days later. Ten healthy, nonsmoking adults were asked, via questionnaire after each day's use, whether they would use nasal high flow therapy while being unwell with a cold or flu if it was demonstrated to improve symptoms. Results. All participants completed both interventions. Eighty percent responded “yes” to future use of nasal high flow therapy, for both 37°C and 41°C. There was no significant change from baseline in saccharin times following either intervention or in the following morning. Conclusions. Delivering humidified air via nasal high flow therapy at both 37°C and 41°C is well tolerated by healthy adults. This supports investigation into the potential use of nasal high flow therapy as treatment in viral upper respiratory tract infections. Trial Registration. This trial is registered with ACTRN12614000183684 (tolerability study of nasal delivery of humidified & warmed air).

Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts

Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts.

Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.

Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells.

The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle. The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.

Attenuation of the influenza virus by microRNA response element in vivo and protective efficacy against 2009 pandemic H1N1 virus in mice

The 2009 influenza pandemics underscored the need for effective vaccines to block the spread of influenza virus infection. Most live attenuated vaccines utilize cold-adapted, temperature-sensitive virus. An alternative to live attenuated virus is presented here, based on microRNA-induced gene silencing. In this study, miR-let-7b target sequences were inserted into the H1N1 genome to engineer a recombinant virus - miRT-H1N1. Female BALB/c mice were vaccinated intranasally with the miRT-H1N1 and challenged with a lethal dose of homologous virus. This miRT-H1N1 virus was attenuated in mice, while it exhibited wild-type characteristics in chicken embryos. Mice vaccinated intranasally with the miRT-H1N1 responded with robust immunity that protected the vaccinated mice from a lethal challenge with the wild-type 2009 pandemic H1N1 virus. These results indicate that the influenza virus containing microRNA response elements (MREs) is attenuated in vivo and can be used to design a live attenuated vaccine.

All-in-one bacmids: an efficient reverse genetics strategy for influenza A virus vaccines.

Vaccination is the first line of defense against influenza virus infection, yet influenza vaccine production methods are slow, antiquated, and expensive as a means to effectively reduce the virus burden during epidemic or pandemic periods. There is a great need for alternative influenza vaccines and vaccination methods with a global scale of impact. We demonstrate here a strategy to generate influenza A virus in vivo by using bacmid DNAs. Compared to the classical reverse genetics system, the "eight-in-one" bacmids (bcmd-RGFlu) showed higher efficiency of virus rescue in various cell types. Using a transfection-based inoculation (TBI) system, intranasal delivery to DBA/2J and BALB/c mice of bcmd-RGFlu plus 293T cells led to the generation of lethal PR8 virus in vivo. A prime-boost intranasal vaccination strategy using TBI in the context of a bcmd-RGFlu carrying a temperature-sensitive H1N1 virus resulted in protection of mice against lethal challenge with the PR8 strain. Taken together, these studies provide proof of principle to highlight the potential of vaccination against influenza virus by using in vivo reverse genetics. Vaccination is the first line of defense against influenza virus infections. A major drawback in the preparation of influenza vaccines is that production relies on a heavily time-consuming process of growing the viruses in eggs. We propose a radical change in the way influenza vaccination is approached, in which a recombinant bacmid, a shuttle vector that can be propagated in both Escherichia coli and insect cells, carries an influenza virus infectious clone (bcmd-RGFlu). Using a surrogate cell system, we found that intranasal delivery of bcmd-RGFlu resulted in generation of influenza virus in mice. Furthermore, mice vaccinated with this system were protected against lethal influenza virus challenge. The study serves as a proof of principle of a potentially universal vaccine platform against influenza virus and other pathogens.

Effects of weather variables on thermoregulation of calves during periods of extreme heat

To determine effects of ambient temperature, relative humidity, wind speed, relative barometric pressure, and temperature-humidity index (THI) on nasal submucosal and rectal temperatures in cattle during extreme summer conditions. 20 black crossbred beef heifers (mean body weight, 217.8 kg). Nasal submucosal and rectal temperatures were monitored every 2 hours for 24 hours on 3 nonconsecutive days when ambient temperature was forecasted to exceed 32.2°C. Ambient temperature, relative humidity, wind speed, and relative barometric pressure were continuously monitored at a remote weather station located at the research facility. The THI was calculated and used in the livestock weather safety index (LWSI). Relationships between nasal submucosal or rectal temperature and weather variables were evaluated. Nasal submucosal and rectal temperatures were related to all weather variables monitored. A positive relationship was determined for ambient temperature and THI with both nasal submucosal and rectal temperatures. A negative relationship was evident for nasal submucosal and rectal temperature with relative humidity, wind speed, and relative barometric pressure. Nasal submucosal and rectal temperatures increased with increasing severity of LWSI category. Effects of environmental conditions on thermoregulation in calves exposed to extreme heat were detected. The positive relationship between nasal submucosal temperature and ambient temperature and THI raised concerns about the efficacy of intranasal administration of temperature-sensitive modified-live virus vaccines during periods of extreme heat. Environmental conditions must be considered when rectal temperature is used as a diagnostic tool for identifying morbid cattle.