Accessing Genes from Environmental DNA Libraries

The classical approach for isolating enzymes from environmental samples is to enrich, isolate and screen a variety of microorganisms for the desired enzyme activity. The majority of the physiological diversity is excluded with this approach because it is estimated that >99% of the microorganisms observed in the environment cannot be cultivated. An alternative to this cultivation-dependent approach is to clone DNA which has been extracted directly from microbial biomass present in water, soil and sediment. Using an appropriate host system, enzyme activity can subsequently be detected by screening for heterologous gene expression. Geothermal regions are sources of thermophilic microbial diversity. This study sought to investigate the methods for extracting and cloning DNA from geothermal sediments for the purpose of detecting thermostable enzyme activities. Methods for extracting and purifying DNA directly from soil and sediment were evaluated based on DNA yield, purity and quality. Purified environmental DNA was then used to evaluate cloning protocols based on cloning efficiency, recombination efficiency and total number of recombinants generated per ligation reaction. Subsequently, two environmental gene libraries were constructed using a TA-cloning method with DNA directly extracted from sediments that were collected from Iceland geothermal sites. The environmental library designated as ICE16 was derived from biomass present in sediment at -74°C, pH 7.4, while the DNA used to construct library ICE22 was derived from biomass present in sediment at -58°C, pH 4.3. These libraries were screened for thermostable amylase, lipase, protease and phosphatase activities using established assays in both microtitre plate and indicator agar-plate formats. One transformant possessing phosphatase activity at 60°C (Phos22) and two transformants showing phenotypic differences on starch agar plates at 50°C (5ICE16, 6ICE16) were recovered from the ICE22 and ICE16 DNA libraries, respectively. These clones were selected for further evaluation including sequencing and expression studies

Recruitment of Cellular Recombination and Repair Proteins to Sites of Herpes Simplex Virus Type 1 DNA Replication Is Dependent on the Composition of Viral Proteins within Prereplicative Sites and Correlates with the Induction of the DNA Damage Response

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10

Inhibition of the Herpes Simplex Virus Type 1 DNA Polymerase Induces Hyperphosphorylation of Replication Protein A and Its Accumulation at S-Phase-Specific Sites of DNA Damage during Infection

The treatment of mammalian cells with genotoxic substances can trigger DNA damage responses that include the hyperphosphorylation of replication protein A (RPA), a protein that plays key roles in the recognition, signaling, and repair of damaged DNA. We have previously reported that in the presence of a viral polymerase inhibitor, herpes simplex virus type 1 (HSV-1) infection induces the hyperphosphorylation of RPA (D. E. Wilkinson and S. K. Weller, J. Virol. 78:4783-4796, 2004). We initiated the present study to further characterize this genotoxic response to HSV-1 infection. Here we report that infection in the presence of polymerase inhibitors triggers an S-phase-specific response to DNA damage, as demonstrated by induction of the hyperphosphorylation of RPA and its accumulation within viral foci specific to the S phase of the cell cycle. This DNA damage response occurred in the presence of viral polymerase inhibitors and required the HSV-1 polymerase holoenzyme as well as the viral single-stranded-DNA binding protein. Treatment with an inhibitor of the viral helicase-primase did not induce the hyperphosphorylation of RPA or its accumulation in infected cells. Taken together, these results suggest that the S-phase-specific DNA damage response to infection is dependent on the specific inhibition of the polymerase. Finally, RPA hyperphosphorylation was not induced during productive infection, indicating that active viral replication does not trigger this potentially detrimental stress response

Oligomerization of ICP4 and Rearrangement of Heat Shock Proteins May Be Important for Herpes Simplex Virus Type 1 Prereplicative Site Formation▿

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen

AbstractBackgroundThe WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012.ObjectivePreparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU).Study designThe 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B.ResultsTwelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use.Conclusions12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays

Establishment of the 1st World Health Organization international standards for human papillomavirus type 16 DNA and type 18 DNA

A World Health Organization collaborative study was conducted to evaluate candidate international standards for human papillomavirus (HPV) Type 16 DNA (NIBSC code 06/202) and HPV Type 18 DNA (NIBSC code 06/206) for use in the amplification and detection steps of nucleic acid-based assays. The freeze-dried candidate international standards were prepared from bulk preparations of cloned plasmid containing full-length HPV-16 or HPV-18 genomic DNA. Nineteen laboratories from 13 countries participated in the study using a variety of commercial and in-house quantitative and qualitative assays. The data presented here indicate that, upon freeze-drying, there is no significant loss in potency for the candidate HPV-18 DNA and a slight loss in potency for the candidate HPV-16 DNA; although this is likely not scientifically relevant when assay precision is considered. In general, the individual laboratory mean estimates for each study sample were grouped ± ˜2 log10 around the theoretical HPV DNA concentration of the reconstituted ampoule (1 × 107 HPV genome equivalents/mL). The agreement between laboratories is improved when potencies are made relative to the candidate international standards, demonstrating their utility in harmonizing amplification and detection steps of HPV-16 and -18 DNA assays. Degradation studies indicate that the candidate international standards are extremely stable and suitable for long-term use. Based on these findings, the candidate standards were established as the 1st WHO international standards for HPV-16 DNA and HPV-18 DNA, each with a potency of 5 × 106 international units (IU) per ampoule or 1 × 107 IU mL-1 when reconstituted as directed