Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene
|
|
- Jemima Douglas
- 6 years ago
- Views:
Transcription
1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2005, p Vol. 49, No /05/$ doi: /aac Copyright 2005, American Society for Microbiology. All Rights Reserved. Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene Sunwen Chou,* Laura C. Van Wechel, Heather M. Lichy, and Gail I. Marousek Medical and Research Services, VA Medical Center and Oregon Health & Science University, Portland, Oregon Received 30 November 2004/Returned for modification 8 February 2005/Accepted 3 March 2005 A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC 50 ). Changes in the IC 50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate. Treatment of cytomegalovirus (CMV) infections with current antiviral drugs ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) may select for viral drug resistance mutations. CMV UL97 phosphotransferase gene mutations at codons 460, 520, and 590 to 607 have been shown to confer ganciclovir resistance (1, 4), while an increasingly diverse set of viral UL54 DNA polymerase (pol) mutations confer varying degrees of resistance and cross-resistance to the three drugs (2, 3). Resistance of CMV to antiviral drugs may be diagnosed genotypically, by looking for known viral resistance mutations in UL97 or pol, or phenotypically, by applying various drug concentrations to a growing virus in cell culture. Genotypic analysis is rapid and can be performed without the need for viral isolation, but mutations of indeterminate significance for resistance continue to be reported without phenotypic validation (8) and the degree of resistance conferred by combinations of mutations may be difficult to deduce by genotypic testing alone. Phenotypic analysis detects drug resistance without the need for genetic information, but even if a CMV isolate is available for testing, the standard plaque reduction assay is slow, technically demanding, and shows a high degree of interlaboratory variability (6). For diagnosis of CMV drug resistance in a clinical setting, genotypic methods are more practical, but this requires the continuing study of phenotypes corresponding to new mutations encountered in clinical isolates. The drug resistance phenotype corresponding to specific viral mutations can be determined by transferring them to a known drug-sensitive reference strain and testing for the resulting change in drug sensitivity. In the past few years this process, known as marker transfer or recombinant phenotyping, has been facilitated by the development of CMV strains * Corresponding author. Mailing address: VA Medical Center, P3ID 3710 SW U.S. Veterans Hospital Road, Portland, OR Phone: (503) Fax: (503) chous@ohsu.edu. containing unique restriction sites in UL97 or pol that permit the rapid generation of recombinant viruses containing desired mutations (2, 4). Phenotyping of the resulting recombinant viruses then becomes the rate-limiting step, especially when performed by the standard plaque reduction assay. Here, we report the construction of CMV strains containing unique restriction sites in UL97 and pol, along with a secreted alkaline phosphatase (SEAP) reporter gene inserted at the nonessential gene region US6. This enabled the generation of recombinant viruses containing desired mutations in either or both UL97 and pol, which could be quantitated by simply measuring the SEAP activity in the culture supernatant. Reduction in SEAP activity under drug was used to determine drug sensitivity. Results of transferring specific mutations were compared with results previously obtained using traditional phenotyping assays, and the effects of combinations of mutations were studied for the first time. (Presented in part at the Infectious Diseases Society of America Annual Meeting, Boston, Mass., October 2004 [abstract 641] and the 44th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, October 2004 [abstract 615].) MATERIALS AND METHODS Viruses and plasmid transfer vectors. CMV strain AD169 (ATCC VR-538) was obtained from the American Type Culture Collection. All CMV strains and recombinants were propagated in human foreskin fibroblasts. Mutagenesis in the viral UL97 phosphotransferase and UL54 DNA polymerase (pol) genes was accomplished by homologous recombination using plasmid transfer vectors analogous to those previously published (2, 4). In the UL97 region, the starting plasmid was a Bluescript subclone of the AD169 genome (GenBank X17403) from nucleotides to (NotI and KpnI restriction sites, respectively). In the pol region, the corresponding subclone contained AD169 nucleotides to (XbaI and EcoRI restriction sites, respectively). Desired nucleotide changes (unique restriction sites PmeI or SwaI, or resistance-related mutations) were introduced into these transfer vectors by replacement of segments of these subclones by PCR products as described elsewhere (2, 4). A green fluorescent protein selectable marker (hrgfp) or SEAP reporter 2710
2 VOL. 49, 2005 CMV RECOMBINANT PHENOTYPING 2711 Recombinant strain Parental strain (genomic DNA) TABLE 1. Genotypes of AD169-derived recombinant CMV strains Genotype UL97 pol US6 region Selection marker T1421 AD169 wt a PmeI (453), PmeI (S897L) P522S V7811 wt GCVr FOSr T1937 T1421 wt PmeI (453), PmeI (S897L) wt None T2025 T1937 SwaI (H587Y) L595S PmeI (453), PmeI (S897L) wt GCVr T2065 T2025 SwaI (H587Y) PmeI (453), PmeI (S897L) MIE-hrGFP GFP T2211 T2065 SwaI (H587Y) PmeI (453), PmeI (S897L) MIE-SEAP None a wt, wild type. gene, both driven by CMV major immediate-early (MIE) promoters, were targeted for insertion at CMV US6 by flanking the expression cassettes with 2 kb of US3 sequence on one side and of US6 sequence on the other side, each incorporating PacI restriction sites at the junctions. This region of the CMV genome is dispensable for normal viral replication in cell culture. The hrgfp expression vector (MP234) was obtained from Mark Peeples (Rush Presbyterian St. Lukes Medical Center, Chicago, Ill.). The SEAP expression vector gwizseap was obtained from Gene Therapy Systems (San Diego, CA). Construction of reference strain T2211. Strain T2211, containing unique PmeI sites in pol, a SwaI site in UL97, and a SEAP expression cassette at US6, was constructed by successive recombination steps producing intermediate viral strains as shown in Table 1. Strain AD169 genomic DNA was extracted from infected fibroblast cultures as previously described (4). The AD169 sequence (GenBank X17403) contains no restriction sites for enzymes PacI, PmeI, or SwaI. To introduce PmeI restriction sites at codons 453 and 897 of the pol gene, a transfer vector containing PmeI sites at those codons and drug resistance markers P522S and V781I was prepared and cotransfected with strain AD169 DNA, followed by selection for recombinant virus T1421 using GCV and FOS, as described previously (2). The drug resistance markers were then removed from T1421 by another round of recombination (2). In the next round, a unique SwaI site was inserted at codons 584 to 586 of UL97 using the same approach as described elsewhere (4) of cotransfection with a GCV resistance marker, L595S, and its subsequent removal. Simultaneously, an hrgfp expression cassette was inserted at US6 by cotransfection and homologous recombination, and fluorescent cells infected with recombinant virus (T2065) were selected by fluorescenceactivated cell sorting. Finally, strain T2065 was plaque purified and its genomic DNA was extracted, digested with PacI, and cotransfected with the SEAP expression cassette targeted at the US6. The resulting nonfluorescent recombinant virus (T2211) (Table 1) was triply plaque purified, and its genotype was verified by PCR amplification and sequencing of the UL97, pol, and US3-US6 gene regions. Strain T2211 was then used as the reference virus for phenotyping and for construction of additional recombinant viruses containing mutations of interest (marker transfer). Marker transfer. Resistance-related mutations in CMV UL97 or pol were transferred into T2211 by cotransfection of T2211 DNA digested with SwaI or PmeI, respectively, and a transfer vector incorporating the desired mutation in the desired gene region. Recombinant virus that grew (usually in 10 to 14 days) was screened by PCR and restriction analysis or DNA sequencing for the desired mutation and was plaque purified thereafter, all without using any drug selection in cell culture. The plaque-purified recombinant viruses were sequenced in the entire coding sequence of the modified genes to confirm the presence of the desired mutations and absence of extraneous ones. Sequencing was performed by dye terminator chemistry (BigDye v1.1; Applied Biosystems) on the ABI377 automated sequencer. Phenotypic assays of recombinant viruses. Cell-free virus stock was prepared from each CMV strain and titrated for infectivity by enumeration of cells staining positive at 24 h for CMV MIE antigen (2). Growth curves were performed at a high and low multiplicities of infection (MOI) to compare the increase of viral infectivity of strains T2211 and AD169. Over 4- and 7-day periods, daily aliquots of culture supernatant were collected and stored at 80 C and the removed medium was replaced with fresh medium. The stored aliquots were then assayed for infectivity by inoculation on fibroblast monolayers and MIE antigen staining (2). SEAP assays were performed by diluting culture supernatant 1:10 (or 1:4 for samples collected the day after inoculation) in dilution buffer (50 mm Tris [ph 7.4], 150 mm NaCl) and heating to 65 C for 30 min. Thirty microliters of this diluted supernatant was then mixed with an equal volume of assay buffer (2 M diethanolamine, ph 9.8, and 28 mm L-homoarginine; both from Sigma), followed 5 min later by 30 l of ready-made chemiluminescent substrate mixture (0.4 mm CSPD with Emerald II enhancer in 0.1 M diethanolamine; Applied Biosystems Tropix). After 15 min, chemiluminescence (in relative light units [RLU]) was read in a 96-well plate luminometer (Glo-Runner; Turner Biosystems). All RLU data here are reported as the luminescence per 30 l of undiluted culture supernatant. Yield reduction assays for drug resistance were performed by inoculating 6 wells of a 24-well culture of human foreskin fibroblasts that were confluent for 3 days with 0.3 ml of virus to be tested, at an MOI of 0.01 to 0.03, corresponding to a SEAP RLU of 400 to 1,500 at 24 h (see Results). After 90 min, the inoculum was removed and the culture medium was then replaced in the wells, five of which contained twofold serial dilutions of drug (GCV, FOS, or CDV) above and below typical inhibitory concentrations (see Results). Four to 7 days after inoculation, aliquots of culture supernatant from wells with and without drug were assayed for SEAP activity as described above. The drug concentration (IC 50 ) required to reduce the SEAP activity to 50% of the control value (in the well without added drug) was calculated by fitting an exponential curve to the SEAP activities measured in the wells containing various drug concentrations. The IC 50 values of at least five separate experiments with each drug and virus were averaged, and results are reported as mean and standard deviation values. The ratio of IC 50 value to that of a sensitive control strain assayed at the same postinoculation interval was used as an index of drug sensitivity and was compared to values in the existing literature for the mutations being studied. RESULTS Properties of CMV strain T2211. Strain T2211 was derived from strain AD169 after several recombination steps, as listed in Table 1, resulting in the genotype shown. The insertions of unique restriction sites in UL97 and pol were accompanied by single amino acid changes at codons 587 and 897, respectively. T2211 also contained a SEAP gene cassette at US6 driven by a copy of the MIE promoter (Fig. 1). Despite these genetic changes, strain T2211 had the same cytopathic effect in culture, and similar infectivity growth curves at both high and low MOI, as the parental strain, AD169 (Fig. 2). Strain T2211 appeared to be slightly slower growing, but this did not affect the ability to prepare high-titer cell-free virus stock or obtain normal yields of genomic viral DNA for transfection. SEAP activity was readily detectable in culture supernatants of strain T2211 and its derivatives at 24 h postinoculation, and the activity as measured by chemiluminescence (in RLU) at this time point was directly related to the MOI (Fig. 3). After infection at an MOI of 0.01 to 0.03, accumulated SEAP activity in the culture supernatant increased to 30,000-fold over baseline at 6 days and reached a plateau shortly thereafter. At days 4 to 6, the rate of increase in SEAP activity was comparable to that of viral infectivity (Fig. 2). The growth kinetics of T2211 under different concentrations of GCV, FOS, and CDV were used to determine the optimum day of assay of SEAP activity for purposes of drug sensitivity phenotyping. Based on the shape of the growth curves (Fig. 4), days 5 or 6 postinoculation appeared to be the best times for determining IC 50
3 2712 CHOU ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Genome map of recombinant CMV strain T2211. This strain was derived in several steps from strain AD169 (Table 1) and incorporates unique restriction sites in the viral pol and UL97 gene regions, as well as a SEAP reporter gene at US6 driven by the CMV major immediate-early promoter. values because virus (SEAP activity) was still growing exponentially both with and without drug. For reproducibility of IC 50 values, the MOI was kept between 0.01 and 0.03 as judged by the SEAP activity at 24 h (400 to 1,500 RLU) (Fig. 3). Marker transfer (recombinant phenotyping). New recombinant strains containing desired sequence changes in UL97 or pol were created by digestion of T2211 DNA followed by homologous recombination. Control strains were derived to show the phenotypic effects of amino acid changes in UL97 and pol resulting from insertion of the restriction sites in strain T2211. The wild-type amino acid sequences in UL97 and pol were restored in strains T2233 and T2241, respectively. The drug sensitivity phenotypes for all three drugs, GCV, FOS, and CDV, were not significantly different for strains T2211, T2233, and T2241 (Table 2), indicating a lack of phenotypic effect from the amino acid changes in UL97 and pol in strain T2211. The sensitive strains showed IC 50 values for GCV, FOS, and CDV (Table 2) that were comparable to those reported with plaque reduction, although the latter assay showed very wide interlaboratory variation (6). For example, published strain AD169 plaque reduction IC 50 values (6) include, for GCV, 1.0 to 9.6 M (mean, 4.0) and, for FOS, 35 to 112 M (mean, 62). Other phenotyping assays may give somewhat different results; the DNA hybridization method used for some of the historical phenotypes in Table 2 gave a lower AD169 GCV IC 50 value of 0.55 to 0.74 M (1). Based on the standard deviations of the IC 50 determinations (Table 2), mutations conferring twofold or greater changes in IC 50 values should be reliably identified. The most common UL97 GCV resistance mutations (1, 4) were transferred into T2211, creating the recombinant strains listed in Table 2. Yield reduction IC 50 values showed the resistance mutations M460V, A594V, and L595S to confer 7- to 10-fold increases in the IC 50 for GCV, similar to what had previously been reported for these mutations (1), and the mutation C592G to confer a more modest 2.5- to 3-fold resistance to GCV, as previously reported for a plaque reduction assay (4). The D605E change is the only reported sequence polymorphism found in baseline sensitive CMV isolates in the UL97 codon range 590 to 607, where GCV resistance mutations are FIG. 2. Growth curves of CMV strains AD169 and T2211. Cultures were inoculated at the indicated MOI, and the culture supernatants were sampled at intervals for the amount of infectious virus. Day zero values represent the infectivity of the input virus. In parallel, SEAP activity (RLU) in the supernatant was measured for T2211 cultured at an MOI of Each data point and error bar represents the mean standard error of the mean of three experiments.
4 VOL. 49, 2005 CMV RECOMBINANT PHENOTYPING 2713 clustered (7). As expected, the transfer of this amino acid change (strain T2278) did not confer any change in GCV sensitivity (Table 2). It was reported that the D605E polymorphism reversed the GCV resistance conferred by the mutation A594P in an expressed enzyme assay system (5). To examine whether D605E affected the phenotype conferred by the four most common UL97 resistance mutations, we transferred D605E in combination with each of the mutations, L595S, A594V, M460V, and C592G. Results (Table 2) showed that the differences in IC 50 values for the mutations with and without D605E did not exceed the standard errors of the assays. A known pol resistance mutation (deletion of codon or del981-2) was chosen for transfer into T2211 because it confers resistance to all three drugs, GCV, FOS, and CDV (3). This resulted in strain T2222. As shown in Table 2, the triple resistance phenotype as measured by SEAP IC 50 values for the pol del981-2 mutation was quantitatively similar to that previously determined by plaque reduction (3). To demonstrate the effect of combined UL97 and pol mutations, a double mutant strain T2261 was derived from T2222 by digesting its genomic DNA with SwaI and recombination with a transfer vector containing UL97 C592G. The double mutant containing UL97 mutation C592G and pol mutation del981-2 showed much higher GCV resistance (Table 2) than strains containing either mutation alone (T2222 or T2258). DISCUSSION FIG. 3. Multiplicity of infection versus SEAP activity at 24 h. Cell cultures were inoculated at various multiplicities of infection up to 0.04, and the SEAP activity in culture supernatants was assayed at 24 h postinoculation. Uninoculated cell culture supernatants had a background RLU reading of 25. The data points were fit to a logarithmic curve, giving the correlation coefficient (R 2 ) shown. Construction of a reference CMV laboratory strain T2211 containing unique restriction sites, together with a SEAP reporter gene, enabled the efficient recombinant phenotyping of mutations in the CMV UL97 and pol genes associated with drug resistance. This technical approach was validated by transfer of the most common UL97 GCV resistance mutations and a pol mutation conferring multidrug resistance. Extending previous work, the synergistic effect of combined UL97 and pol mutations on GCV resistance is now proven by marker transfer, and the status of UL97 D605E as a sequence polymorphism without effect on GCV resistance has also been demonstrated. For the known resistance mutations transferred into strain T2211, the amounts (ratios) by which the SEAP yield reduction IC 50 values increased over a sensitive control strain are closely comparable to values previously reported for the same mutations. The absolute IC 50 values vary because of differences across assays and laboratories, but most of the common UL97 mutations observed in clinical isolates have been reported to confer a 5- to 10-fold increase in GCV resistance, while some others confer only a borderline 2- to 3-fold increase (4). With the current SEAP-based phenotyping system, it is confirmed that the UL97 mutation C592G confers a lesser degree of GCV resistance than M460V, A594V, and L595S and that the pol mutation del981-2 confers multidrug resistance. Although the UL97 C592G mutation and the pol del981-2 mutation individually confer only modest GCV resistance, the combination of the two confers a much higher level of resistance, as previously hypothesized but not proven by marker transfer (9). FIG. 4. SEAP growth curves of CMV strain T2211 under GCV, FOS, and CDV. Cell cultures were inoculated at an MO1 of 0.01 and grown under various drug concentrations (A GCV, B FOS, and C CDV). SEAP activity in culture supernatants was assayed daily at 4 to 7 days postinoculation. Each data point and error bar is the mean standard error of the mean of eight experiments.
5 2714 CHOU ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Genotypes and phenotypes of T2211-derived CMV strains CMV strain Genotype a IC 50 (ratio) c by SEAP assay b Published marker transfer d (ratio) c UL97 pol GCV FOS CDV GCV FOS CDV Controls T2211 SwaI (H587Y) PmeI (S897L) T2233 wt e PmeI (S897L) T2241 SwaI (H587Y) wt T2278 D605E PmeI (S897L) Mutants T2259 M460V PmeI (S897L) (8.3) 5.2 (7.0) T2256 M460V D605E PmeI (S897L) (9.8) T2258 C592G PmeI (S897L) (2.9) 45 5 (0.9) (1.2) 14.5 (2.6) T2247 C592G D605E PmeI (S897L) (2.6) T2255 A594V PmeI (S897L) (8.3) 5.9 (10.7) T2235 A594V D605E PmeI (S897L) (7.7) T2260 L595S PmeI (S897L) (9.2) 6.4 (11.6) T2234 L595S D605E PmeI (S897L) (8.5) T2222 SwaI (H587Y) del( ) (4.8) (3.5) (4.1) 47 (6.1) 268 (4.0) 1.8 (5.1) T2261 C592G del( ) (16.9) (4.1) (5.7) a Resistance mutations are shown in bold typeface. b IC 50 values are in M, expressed as mean standard deviation of at least five assays. c Ratio of IC 50 to that of sensitive control strain. d Previously published IC 50 and ratio for the same resistance mutations (1, 3, 4). e wt, wild type. The known UL97 GCV resistance mutations are tightly clustered at codons 460, 520, and the range of 590 to 607 (4). Most of the assorted amino acid changes (including deletions) that have been observed in CMV isolates at codons 590 to 607 are associated with varying degrees of GCV resistance (4), with the notable exception of D605E (7). Based on studies of recombinant vaccinia viruses expressing UL97, the D605E change was proposed to reverse the GCV resistance conferred by the unusual mutation A594P (5). Here, after examining the phenotypes of recombinant viruses containing the most common UL97 mutations with and without D605E, we confirm that D605E does not confer any GCV resistance, but we cannot confirm that the polymorphism has a significant effect on the degree of resistance conferred by the most common UL97 resistance mutations. It is possible that the effect of D605E is too small to measure in our assay system or that it occurs with only certain less-common UL97 mutations. The major advantages of this recombinant phenotyping approach are its relative rapidity and reproducibility, along with the ability to study combinations of UL97 and pol mutations. The entire process (several weeks) can actually be faster than propagating, quantitating, and testing a clinical isolate by conventional plaque reduction. Compared with clinical isolates, the high-titer extracellular virus produced by the laboratory strains and recombinants simplifies quantitative work. Because the SEAP reporter signal is measured directly from culture supernatants with minimal processing, fewer variables are introduced during the quantitation step. Use of multichannel equipment and a 96-well plate luminometer greatly increases throughput. These features permit the proper control of assay variables, such as the viral inoculum or culture duration, and allow the generation of sufficient replicate assays to assess the reproducibility of the results. Cultures may be nondestructively sampled at several time points to assess growth characteristics and optimize the timing of the assay. The various CMV phenotyping assays in current use measure different aspects of virus growth, and comparability of results must be empirically determined. Readouts of viral growth have been based on plaque formation, extracted viral DNA, extracted viral antigen, or antigen detection in infected cells (including flow cytometry). The current assay measures the activity of the CMV MIE promoter that drives the SEAP reporter gene. This promoter is most active early in the viral replication cycle, and the exponential increase in SEAP activity during multiple cycles of growth reflects the infection of new cells as well as the stability of the secreted enzyme. So far, we have validated this multicycle SEAP-based assay only for the current CMV drugs (GCV, FOS, and CDV), which all target the CMV DNA polymerase. Extension of this system to experimental CMV drugs with different mechanisms of action will require further study. Use of strain T2211 as a base for transfer of resistance mutations will be useful in studying new mutations, combinations of mutations, and clinical CMV sequences without an available viral isolate. With the adoption of molecular diagnostic methods for CMV, it is now common for the virus to be detected only by direct PCR amplification from clinical specimens, without an accompanying viral isolate. Previously uncharacterized sequence changes (both in UL97 and pol) associated with CMV infection poorly responsive to drug therapy (8) can be assessed more efficiently using this technical approach. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI and by Department of Veterans Affairs research funds. REFERENCES 1. Chou, S., A. Erice, M. C. Jordan, G. M. Vercellotti, K. R. Michels, C. L. Talarico, S. C. Stanat, and K. K. Biron Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and iden-
6 VOL. 49, 2005 CMV RECOMBINANT PHENOTYPING 2715 tification of mutations conferring ganciclovir resistance. J. Infect. Dis. 171: Chou, S., N. S. Lurain, K. D. Thompson, R. C. Miner, and W. L. Drew Viral DNA polymerase mutations associated with drug resistance in human cytomegalovirus. J. Infect. Dis. 188: Chou, S., R. C. Miner, and W. L. Drew A deletion mutation in region V of the cytomegalovirus DNA polymerase sequence confers multidrug resistance. J. Infect. Dis. 182: Chou, S., R. H. Waldemer, A. E. Senters, K. S. Michels, G. W. Kemble, R. C. Miner, and W. L. Drew Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. J. Infect. Dis. 185: Ijichi, O., D. Michel, T. Mertens, K. Miyata, and Y. Eizuru GCV resistance due to the mutation A594P in the cytomegalovirus protein UL97 is partially reconstituted by a second mutation at D605E. Antivir. Res. 53: Landry, M. L., S. Stanat, K. Biron, D. Brambilla, W. Britt, J. Jokela, S. Chou, W. L. Drew, A. Erice, B. Gilliam, N. Lurain, J. Manischewitz, R. Miner, M. Nokta, P. Reichelderfer, S. Spector, A. Weinberg, B. Yen-Lieberman, and C. Crumpacker A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob. Agents Chemother. 44: Lurain, N. S., A. Weinberg, C. S. Crumpacker, and S. Chou Sequencing of the cytomegalovirus UL97 gene for genotypic antiviral resistance testing. Antimicrob. Agents Chemother. 45: Scott, G. M., M. A. Isaacs, F. Zeng, A. M. Kesson, and W. D. Rawlinson Cytomegalovirus antiviral resistance associated with treatment induced UL97 (protein kinase) and UL54 (DNA polymerase) mutations. J. Med. Virol. 74: Smith, I. L., J. M. Cherrington, R. E. Jiles, M. D. Fuller, W. R. Freeman, and S. A. Spector High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J. Infect. Dis. 176:69 77.
Emerging CMV Resistance Profile for CMX001
Emerging CMV Resistance Profile for CMX001 International Conference on Antiviral Research May 15, 2013 Randall Lanier, PhD Forward Looking Statements These slides and the accompanying oral presentation
More informationMutations in the Human Cytomegalovirus UL27 Gene That Confer Resistance to Maribavir
JOURNAL OF VIROLOGY, July 2004, p. 7124 7130 Vol. 78, No. 13 0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.13.7124 7130.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Mutations
More informationAntiviral Drug Resistance of Human Cytomegalovirus
CLINICAL MICROBIOLOGY REVIEWS, Oct. 2010, p. 689 712 Vol. 23, No. 4 0893-8512/10/$12.00 doi:10.1128/cmr.00009-10 Copyright 2010, American Society for Microbiology. All Rights Reserved. Antiviral Drug Resistance
More informationRapid Ganciclovir Susceptibility Assay Using Flow Cytometry for Human Cytomegalovirus Clinical Isolates
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1998, p. 2326 2331 Vol. 42, No. 9 0066-4804/98/$04.00 0 Copyright 1998, American Society for Microbiology. All Rights Reserved. Rapid Ganciclovir Susceptibility
More informationCMV Diagnostic Strategies: Current and Future
CMV Diagnostic Strategies: Current and Future Tony Mazzulli, MD, FRCPC, FACP Microbiologist-in-Chief Mount Sinai Hospital & University Health Network, Toronto Faculty/Presenter Disclosure Relationships
More informationCMV Drug Resistance: Clinical Impact and Potential Strategies Sunwen Chou, MD
CMV Drug Resistance: Clinical Impact and Potential Strategies Sunwen Chou, MD Slide 1 CMV Drug Resistance: Clinical Impact and Potential Strategies Thank you. Slide 2 CMV Resistance Typical Setting As
More informationResistance of Human Cytomegalovirus to Antiviral Drugs
CLINICAL MICROBIOLOGY REVIEWS, Apr. 1999, p. 286 297 Vol. 12, No. 2 0893-8512/99/$04.00 0 Copyright 1999, American Society for Microbiology. All Rights Reserved. Resistance of Human Cytomegalovirus to
More informationAvailable online at
Available online at www.annclinlabsci.org 429 Letter to Editor: Detection of a UL97 Gene Mutation Conferring Ganciclovir Resistance in Human Cytomegalovirus: Prevalence of the D605E Polymorphism in Korean
More informationNature Medicine: doi: /nm.4322
1 2 3 4 5 6 7 8 9 10 11 Supplementary Figure 1. Predicted RNA structure of 3 UTR and sequence alignment of deleted nucleotides. (a) Predicted RNA secondary structure of ZIKV 3 UTR. The stem-loop structure
More informationReceived 9 June 2004/Returned for modification 27 August 2004/Accepted 10 September 2004
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2005, p. 208 213 Vol. 43, No. 1 0095-1137/05/$08.00 0 doi:10.1128/jcm.43.1.208 213.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. How
More informationAnimal hosts Natural host Laboratory animals Rabbits Mice Rats Hamsters Newborn or suckling rodents Animal models for viral pathogenesis 4 Growth of v
Principles of Virology Department of Molecular Genetics & Microbiology Univ ersity of Florida, Gainesv ille, FL 1 Outline Virus cultivation Assay of viruses Virus genetics 2 Virus isolation Evidence of
More informationCRISPRaTest Functional dcas9-activator Assay Kit v1 Last update: 2018/07/04 Cellecta, Inc.
CRISPRaTest Functional dcas9-activator Assay Kit v1 Last update: 2018/07/04 Cellecta, Inc. Copyright (c) 2018 Cellecta, Inc. All Rights Reserved. Table of Contents 1. CRISPRaTest Functional dcas9-activator
More informationSupplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Supplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid. HEK293T
More informationGanciclovir-resistant CMV retinitis in a patient with wild-type plasma CMV. North Carolina School of Medicine, Chapel Hill, NC
JCM Accepts, published online ahead of print on 15 February 2012 J. Clin. Microbiol. doi:10.1128/jcm.00029-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 Ganciclovir-resistant
More informationOriginal article Cytomegalovirus resistance in solid organ transplant recipients treated with intravenous ganciclovir or oral valganciclovir
Antiviral Therapy 14:697 704 Original article Cytomegalovirus resistance in solid organ transplant recipients treated with intravenous ganciclovir or oral valganciclovir Guy Boivin 1,2 *, Nathalie Goyette
More informationAli Alabbadi. Bann. Bann. Dr. Belal
31 Ali Alabbadi Bann Bann Dr. Belal Topics to be discussed in this sheet: Particles-to-PFU Single-step and multi-step growth cycles Multiplicity of infection (MOI) Physical measurements of virus particles
More informationHepatitis B Antiviral Drug Development Multi-Marker Screening Assay
Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Background ImQuest BioSciences has developed and qualified a single-plate method to expedite the screening of antiviral agents against
More informationNEXT GENERATION SEQUENCING OPENS NEW VIEWS ON VIRUS EVOLUTION AND EPIDEMIOLOGY. 16th International WAVLD symposium, 10th OIE Seminar
NEXT GENERATION SEQUENCING OPENS NEW VIEWS ON VIRUS EVOLUTION AND EPIDEMIOLOGY S. Van Borm, I. Monne, D. King and T. Rosseel 16th International WAVLD symposium, 10th OIE Seminar 07.06.2013 Viral livestock
More informationDiagnostic Methods of HBV and HDV infections
Diagnostic Methods of HBV and HDV infections Zohreh Sharifi,ph.D Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Hepatitis B-laboratory diagnosis Detection
More informationMurine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M35 Is Defective in Growth In Vivo
JOURNAL OF VIROLOGY, July 2003, p. 7746 7755 Vol. 77, No. 14 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.14.7746 7755.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Murine Cytomegalovirus
More informationPre-made Reporter Lentivirus for JAK-STAT Signaling Pathway
Pre-made Reporter for JAK-STAT Signaling Pathway Cat# Product Name Amounts LVP937-P or: LVP937-P-PBS ISRE-GFP (Puro) LVP938-P or: LVP938-P-PBS ISRE-RFP (Puro) LVP939-P or: LVP939-P-PBS ISRE-Luc (Puro)
More informationThe Infectious Cycle. Lecture 2 Biology W3310/4310 Virology Spring You know my methods, Watson --SIR ARTHUR CONAN DOYLE
The Infectious Cycle Lecture 2 Biology W3310/4310 Virology Spring 2016 You know my methods, Watson --SIR ARTHUR CONAN DOYLE The Infectious Cycle Virologists divide the infectious cycle into steps to facilitate
More informationPre-made Reporter Lentivirus for NF-κB Signal Pathway
Pre-made Reporter for NF-κB Signal Pathway Cat# Product Name Amounts LVP965-P or: LVP965-P-PBS NFKB-GFP (Puro) LVP966-P or: LVP966-P-PBS NFKB-RFP (Puro) LVP967-P or: LVP967-P-PBS NFKB-Luc (Puro) LVP968-P
More informationPre-made Reporter Lentivirus for MAPK/ERK Signal Pathway
Pre-made Reporter for MAPK/ERK Signal Pathway Cat# Product Name Amounts LVP957-P or: LVP957-P-PBS SRE-GFP (Puro) LVP958-P or: LVP958-P-PBS SRE-RFP (Puro) LVP959-P or: LVP959-P-PBS SRE-Luc (Puro) LVP960-P
More informationClinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness
CLINICAL MICROBIOLOGY REVIEWS, Oct. 2007, p. 550 578 Vol. 20, No. 4 0893-8512/07/$08.00 0 doi:10.1128/cmr.00017-07 Copyright 2007, American Society for Microbiology. All Rights Reserved. Clinical Significance
More informationSupplemental Materials and Methods Plasmids and viruses Quantitative Reverse Transcription PCR Generation of molecular standard for quantitative PCR
Supplemental Materials and Methods Plasmids and viruses To generate pseudotyped viruses, the previously described recombinant plasmids pnl4-3-δnef-gfp or pnl4-3-δ6-drgfp and a vector expressing HIV-1 X4
More informationConditional and reversible disruption of essential herpesvirus protein functions
nature methods Conditional and reversible disruption of essential herpesvirus protein functions Mandy Glaß, Andreas Busche, Karen Wagner, Martin Messerle & Eva Maria Borst Supplementary figures and text:
More informationmodified dye uptake assay including formazan test EC 90 not tested plaque reduction assay
Sauerbrei A, Bohn-Wippert K, Kaspar M, Krumbholz A, Karrasch M, Zell R. 2015. Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes
More informationRetro-X qrt-pcr Titration Kit User Manual
Takara Bio USA Retro-X qrt-pcr Titration Kit User Manual Cat. No. 631453 PT3952-1 (030218) 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: techus@takarabio.com United States/Canada
More informationHuman Cytomegalovirus (HCMV) Immediate early proteins, gene expression and signaling
Viruses, Cells and Disease November 13, 2008 Human Cytomegalovirus (HCMV) Immediate early proteins, gene expression and signaling Dr. Hua Zhu ICPH E350D UMDNJ - New Jersey Medical School 973-972-4483 X
More informationOxford Expression Technologies Ltd
Oxford Expression Technologies Ltd Founded in 2007 as a spin out from Oxford Brookes University and Natural Environment Research Council Technology based on the insect baculovirus expression vectors (BEVs)
More informationRecombinant Phenotyping of Cytomegalovirus UL54 Mutations that. Emerged During Cell Passages in the Presence of Either Ganciclovir or. Foscarnet.
AAC Accepts, published online ahead of print on 27 June 2011 Antimicrob. Agents Chemother. doi:10.1128/aac.00334-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions.
More informationRecombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2011, p. 4019 4027 Vol. 55, No. 9 0066-4804/11/$12.00 doi:10.1128/aac.00334-11 Copyright 2011, American Society for Microbiology. All Rights Reserved. Recombinant
More informationLaboratory diagnosis of congenital infections
Laboratory diagnosis of congenital infections Laboratory diagnosis of HSV Direct staining Tzanck test Immunostaining HSV isolation Serology PCR Tzanck test Cell scrape from base of the lesion smear on
More informationMutants and HBV vaccination. Dr. Ulus Salih Akarca Ege University, Izmir, Turkey
Mutants and HBV vaccination Dr. Ulus Salih Akarca Ege University, Izmir, Turkey Geographic Distribution of Chronic HBV Infection 400 million people are carrier of HBV Leading cause of cirrhosis and HCC
More informationW. L. William Chang* and Peter A. Barry
JOURNAL OF VIROLOGY, May 2003, p. 5073 5083 Vol. 77, No. 9 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.9.5073 5083.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Cloning of
More informationRelative activity (%) SC35M
a 125 Bat (H17N) b 125 A/WSN (H1N1) Relative activity (%) 0 75 50 25 Relative activity (%) 0 75 50 25 0 Pos. Neg. PA PB1 Pos. Neg. NP PA PB1 PB2 0 Pos. Neg. NP PA PB1 PB2 SC35M Bat Supplementary Figure
More informationFigure S1. Generation of inducible PTEN deficient mice and the BMMCs (A) B6.129 Pten loxp/loxp mice were mated with B6.
Figure S1. Generation of inducible PTEN deficient mice and the BMMCs (A) B6.129 Pten loxp/loxp mice were mated with B6.129-Gt(ROSA)26Sor tm1(cre/ert2)tyj /J mice. To induce deletion of the Pten locus,
More informationVariation in the HindlII Restriction Fragments of DNA from the Chinese Tian Tan Strain of Vaccinia Virus
J. gen. irol. (1985), 66, 1819-1823. Printed in Great Britain 1819 Key words: vaccinia virus~vaccine~restriction Jragrnent variation ariation in the Hindl Restriction Fragments of DNA from the Chinese
More informationExon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation
JOURNAL OF VIROLOGY, June 2005, p. 7438 7452 Vol. 79, No. 12 0022-538X/05/$08.00 0 doi:10.1128/jvi.79.12.7438 7452.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. Exon 3 of
More informationCMV DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real- time PCR Assay Set-up
JCM Accepts, published online ahead of print on 11 May 2011 J. Clin. Microbiol. doi:10.1128/jcm.00721-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights
More informationPre-made Lentiviral Particles for Fluorescent Proteins
Pre-made Lentiviral Particles for Fluorescent Proteins Catalog# Product Name Amounts Fluorescent proteins expressed under sucmv promoter: LVP001 LVP001-PBS LVP002 LVP002-PBS LVP011 LVP011-PBS LVP012 LVP012-PBS
More informationNew Small and Versatile Reporter Technologies for Challenging Applications in Virology. Robert Brazas, Ph.D.
New Small and Versatile Reporter Technologies for Challenging Applications in Virology Robert Brazas, Ph.D. Presentation Overview Small size of NanoLuc Luciferase is advantageous Agenda Building a better
More informationQuantitative Assay of Paravaccinia Virus Based
APPrU MICROBIOLOGY, JUly 1972, p. 138-142 Copyright 1972 American Society for Microbiology Vol. 24, No. 1 Printed in U.S.A. Quantitative Assay of Paravaccinia Virus Based on Enumeration of Inclusion-Containing
More informationIdentification of Mutation(s) in. Associated with Neutralization Resistance. Miah Blomquist
Identification of Mutation(s) in the HIV 1 gp41 Subunit Associated with Neutralization Resistance Miah Blomquist What is HIV 1? HIV-1 is an epidemic that affects over 34 million people worldwide. HIV-1
More informationSURVEILLANCE TECHNICAL
CHAPTER 5 SURVEILLANCE TECHNICAL ASPECTS 55 Protect - detect - protect Polio eradication strategies can be summed up as protect and detect protect children against polio by vaccinating them, and detect
More information7.012 Quiz 3 Answers
MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Friday 11/12/04 7.012 Quiz 3 Answers A > 85 B 72-84
More information33VASTVNGATSANNHGEPPS51PADARPR58
Pro-rich region Trans-membrane region 214 246 359 381 UL50 1 397 211SSRTAS216PPPPPR222 NLS CR1 CR2 CR3 CR4 UL53 1 376 11RERRS15ALRS19LLRKRRR25 33VASTVNGATSANNHGEPPS51PADARPR58 FIG S1. UL97 phosphorylation
More informationTransfection of Sf9 cells with recombinant Bacmid DNA
Transposition Bacmid DNA Mini Culturing baculo cells Transfection of Sf9 cells with recombinant Bacmid DNA Amplification of the virus Titration of baculo stocks Testing the expression Transposition 1.
More informationSupplementary Figure 1 Weight and body temperature of ferrets inoculated with
Supplementary Figure 1 Weight and body temperature of ferrets inoculated with A/Anhui/1/2013 (H7N9) influenza virus. (a) Body temperature and (b) weight change of ferrets after intranasal inoculation with
More informationSupporting Information
Supporting Information Yen et al. 10.1073/pnas.1111000108 SI Materials and Methods Cells. Madin Darby canine kidney (MDCK) cells and human embryonic kidney 293T cells were obtained from the American Type
More information~Lentivirus production~
~Lentivirus production~ May 30, 2008 RNAi core R&D group member Lentivirus Production Session Lentivirus!!! Is it health threatening to lab technician? What s so good about this RNAi library? How to produce
More informationLentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression.
Supplementary Figure 1 Lentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression. a, Design for lentiviral combinatorial mirna expression and sensor constructs.
More informationVIRAL TITER COUNTS. The best methods of measuring infectious lentiviral titer
VIRAL TITER COUNTS The best methods of measuring infectious lentiviral titer FLUORESCENCE CYCLES qpcr of Viral RNA SUMMARY Viral vectors are now routinely used for gene transduction in a wide variety of
More informationPersistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization
J. gen. Virol. (199), 70, 341-345. Printed in Great Britain 341 Key words: influenza C virus/interferon/persistent infection Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization
More informationWORLD HEALTH ORGANIZATION. Smallpox eradication: destruction of Variola virus stocks
WORLD HEALTH ORGANIZATION EXECUTIVE BOARD EB111/5 111th Session 23 December 2002 Provisional agenda item 5.3 Smallpox eradication: destruction of Variola virus stocks Report by the Secretariat 1. The WHO
More information5. Influence of multiplicity of infection (MOI) on synchronous baculovirus infections 5.1. Introduction
5. Influence of multiplicity of infection (MOI) on synchronous baculovirus infections 5.1. Introduction The symptoms of baculovirus infection vary greatly between cells in the same cell culture, even if
More informationBecause accurate and reproducible phenotypic susceptibility
BRIEF REPORT: CLINICAL SCIENCE Comparison of the Precision and Sensitivity of the Antivirogram and PhenoSense HIV Drug Susceptibility Assays Jie Zhang, MS,* Soo-Yon Rhee, MS,* Jonathan Taylor, PhD, and
More informationSupplementary information. MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins
Supplementary information inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins Takuya Tada, Yanzhao Zhang, Takayoshi Koyama, Minoru Tobiume, Yasuko Tsunetsugu-Yokota, Shoji
More informationGenotyping of Cytomegalovirus (or Herpes Simplex Virus) for Ganciclovir (Aciclovir) and Foscarnet Resistance (code 40514) Notice of Assessment
Genotyping of Cytomegalovirus (or Herpes Simple Virus) for Ganciclovir (Aciclovir) and Foscarnet Resistance (code 40514) Notice of Assessment June 2013 DISCLAIMER: This document was originally drafted
More informationDisruption of the M2 Gene of Murine Gammaherpesvirus 68 Alters Splenic Latency following Intranasal, but Not Intraperitoneal, Inoculation
JOURNAL OF VIROLOGY, Feb. 2002, p. 1790 1801 Vol. 76, No. 4 0022-538X/02/$04.00 0 DOI: 10.1128/JVI.76.4.1790 1801.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved. Disruption
More informationIntroduction.-Cytopathogenic viruses may lose their cell-destroying capacity
AN INHIBITOR OF VIRAL ACTIVITY APPEARING IN INFECTED CELL CULTURES* BY MONTO Hot AND JOHN F. ENDERS RESEARCH DIVISION OF INFECTIOUS DISEASES, THE CHILDREN'S MEDICAL CENTER, AND THE DEPARTMENT OF BACTERIOLOGY
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1
More informationFor purification of viral DNA and RNA from a wide range of sample materials
QIAamp virus kits For purification of viral DNA and RNA from a wide range of sample materials Automatable on QIAGEN s proven QIAamp Kits set the standard for purification of viral DNA and RNA. QIAamp virus
More informationGenetic Complementation among Poliovirus Mutants Derived
JOURNAL OF VIROLOGY, Dec. 1986, p. 1040-1049 0022-538X/86/121040-10$02.00/0 Copyright C) 1986, American Society for Microbiology Vol. 60, No. 3 Genetic Complementation among Poliovirus Mutants Derived
More informationJumpstart your research with ViraPower Lentiviral Expression Systems
ViraPower Lentiviral Expression Systems Jumpstart your research with ViraPower Lentiviral Expression Systems With ViraPower Lentiviral Systems you can: Efficiently transduce both dividing and non-dividing
More informationMilan, Italy. Received 15 March 2002; returned 22 July 2002; revised 12 September 2002; accepted 27 September 2002
Journal of Antimicrobial Chemotherapy (2003) 51, 135 139 DOI: 10.1093/jac/dkg016 Comparison of levels of HIV-1 resistance to protease inhibitors by recombinant versus conventional virus phenotypic assay
More informationQuantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications
CLINICAL MICROBIOLOGY REVIEWS, July 1998, p. 533 554 Vol. 11, No. 3 0893-8512/98/$04.00 0 Copyright 1998, American Society for Microbiology. All Rights Reserved. Quantitation of Cytomegalovirus: Methodologic
More informationNBP Protocol. Orders: Support: Web: NBP
NBP2-29541 NBP2-29541 Protocol Orders: orders@novusbio.com Support: technical@novusbio.com Web: www.novusbio.com Protocols, Publications, Related Products, Reviews and more: www.novusbio.com/nbp2-29541
More informationIdentification of Microbes Lecture: 12
Diagnostic Microbiology Identification of Microbes Lecture: 12 Electron Microscopy 106 virus particles per ml required for visualization, 50,000-60,000 magnification normally used. Viruses may be detected
More informationINTRODUCTION PRODUCT DESCRIPTION
INTRODUCTION Mycoplasma are known as important contaminants of biological products derived from cell lines in the biopharmaceutical industry affecting every parameter of a cell culture system. Contaminated
More informationSOME PROPERTIES OF ECHO AND COXSACKIE VIRUSES IN TISSUE CULTURE AND VARIATIONS BY HEAT
THE KURUME MEDICAL JOURNAL Vol. 9, No. 1, 1962 SOME PROPERTIES OF ECHO AND COXSACKIE VIRUSES IN TISSUE CULTURE AND VARIATIONS BY HEAT SHIGERU YAMAMATO AND MASAHISA SHINGU Department of Microbiology, Kurume
More informationCMV Resistance Sequencing
Sequencing test codes: 5600, 30721, 30722 category Infectious Disease cpt code: 87910 Reportable Mutations - UL54 (DNA Polymerase) 1 Viral Phenotype Confirmed by Marker Transfer 2 Mutation Cidofovir Foscarnet
More informationNature Medicine: doi: /nm.2109
HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael
More informationChapter 5. Virus isolation and identification of measles and rubella in cell culture
Chapter 5. Virus isolation and identification of measles and rubella in cell culture In this chapter: 5.1. Recommended cell line for measles and rubella virus isolation 5.2. Propagation of Vero/hSLAM cells
More informationSupplementary Figure 1. FACS analysis of cells infected with TY93/H5N1 GFP-627E,
Supplementary Figure 1. FACS analysis of cells infected with TY93/H5N1 GFP-627E, TY93/H5N1 GFP-627K, or the TY93/H5N1 PB2(588-759) virus library. To establish our GFP- FACS screening platform, we compared
More informationDATA SHEET. Provided: 500 µl of 5.6 mm Tris HCl, 4.4 mm Tris base, 0.05% sodium azide 0.1 mm EDTA, 5 mg/liter calf thymus DNA.
Viral Load DNA >> Standard PCR standard 0 Copies Catalog Number: 1122 Lot Number: 150298 Release Category: A Provided: 500 µl of 5.6 mm Tris HCl, 4.4 mm Tris base, 0.05% sodium azide 0.1 mm EDTA, 5 mg/liter
More informationInfluenza virus exploits tunneling nanotubes for cell-to-cell spread
Supplementary Information Influenza virus exploits tunneling nanotubes for cell-to-cell spread Amrita Kumar 1, Jin Hyang Kim 1, Priya Ranjan 1, Maureen G. Metcalfe 2, Weiping Cao 1, Margarita Mishina 1,
More informationSee external label 2 C-8 C 96 tests Chemiluminescence. CMV IgM. Cat # Diluted samples, controls & calibrator 100 µl 30 minutes
DIAGNOSTIC AUTOMATION, INC. 23961 Craftsman Road, Suite D/E/F, Calabasas, CA 91302 Tel: (818) 591-3030 Fax: (818) 591-8383 onestep@rapidtest.com technicalsupport@rapidtest.com www.rapidtest.com See external
More informationTo test the possible source of the HBV infection outside the study family, we searched the Genbank
Supplementary Discussion The source of hepatitis B virus infection To test the possible source of the HBV infection outside the study family, we searched the Genbank and HBV Database (http://hbvdb.ibcp.fr),
More informationDevelopment of a NIST Standard Reference Material for Cytomegalovirus
Development of a NIST Standard Reference Material for Cytomegalovirus Marcia Holden, Ross Haynes, Margaret Kline, John Butler (with help from David Duewer (NIST) and Steve Ellison (LGC)) Group, Biochemical
More informationReceived 19 May 2004/Accepted 13 September 2004
JOURNAL OF VIROLOGY, Feb. 2005, p. 1666 1677 Vol. 79, No. 3 0022-538X/05/$08.00 0 doi:10.1128/jvi.79.3.1666 1677.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. Genetic Recombination
More informationConstitutive Reporter Lentiviral Vectors Expressing Fluorescent Proteins
Constitutive Reporter Lentiviral Vectors Expressing Fluorescent Proteins www.vectalys.com/products/ Constitutive Reporter Lentiviral Vectors Catalog Number referring to this User Manual: 0008VCT; 0009VCT;
More informationTechnical Bulletin No. 162
CPAL Central Pennsylvania Alliance Laboratory Technical Bulletin No. 162 cobas 6800 HCV Viral Load Assay - New Platform - June 1, 2017 Contact: Heather Habig, MLS (ASCP) CM, MB CM, 717-851-1422 Operations
More informationHIV replication and selection of resistance: basic principles
HIV replication and selection of resistance: basic principles 26th International HIV Drug Resistance and Treatment Strategies Workshop Douglas Richman 6 November 2017 CLINICAL DATA DURING SIXTEEN WEEKS
More informationProblem Set 5 KEY
2006 7.012 Problem Set 5 KEY ** Due before 5 PM on THURSDAY, November 9, 2006. ** Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. 1. You are studying the development
More informationAntiviral-resistant B viruses with a novel mutation detected by antiviral resistance surveillance in Japan
6thMeeting of NICs in the WPR and SEAR 29-31 May, 2012 Hanoi VN Antiviral-resistant B viruses with a novel mutation detected by antiviral resistance surveillance in Japan Masaki Imai WHO Collaborating
More informationAntiviral Chemotherapy
12 Antiviral Chemotherapy Why antiviral drugs? Vaccines have provided considerable success in preventing viral diseases; However, they have modest or often no therapeutic effect for individuals who are
More informationQS S Assist KINASE_ADP-Glo TM Kit
QS S Assist KINASE_ADP-Glo TM Kit Description KINASE ADP-Glo TM kit is designed for use in biochemical kinase assays based on a Luminescent ADP Detection Assay (ADP-Glo TM ). The kit includes human kinase,
More informationRole of the VP16-Binding Domain of vhs in Viral Growth, Host Shutoff Activity, and Pathogenesis
JOURNAL OF VIROLOGY, Dec. 2004, p. 13562 13572 Vol. 78, No. 24 0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.24.13562 13572.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Role
More informationSynthetic Genomics and Its Application to Viral Infectious Diseases. Timothy Stockwell (JCVI) David Wentworth (JCVI)
Synthetic Genomics and Its Application to Viral Infectious Diseases Timothy Stockwell (JCVI) David Wentworth (JCVI) Outline Using informatics to predict drift (strain selection) Synthetic Genomics: Preparedness
More informationConstruction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation
Construction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation J. Du 1, Z.H. Tao 2, J. Li 2, Y.K. Liu 3 and L. Gan 2 1 Department of Chemistry,
More informationEVALUATION OF THE EFFECTIVENESS OF A 7% ACCELERATED HYDROGEN PEROXIDE-BASED FORMULATION AGAINST CANINE PARVOVIRUS
Final report submitted to Virox Technologies, Inc. EVALUATION OF THE EFFECTIVENESS OF A 7% ACCELERATED HYDROGEN PEROXIDE-BASED FORMULATION AGAINST CANINE PARVOVIRUS Syed A. Sattar, M.Sc., Dip. Bact., M.S.,
More informationDiagnostic Methods of HBV infection. Zohreh Sharifi,ph.D of Virology Research center, Iranian Blood Transfusion Organization (IBTO)
Diagnostic Methods of HBV infection Zohreh Sharifi,ph.D of Virology Research center, Iranian Blood Transfusion Organization (IBTO) Hepatitis B-laboratory diagnosis Detection of HBV infection involves
More informationANALYSIS OF MYCOPLASMA GENITALIUM STRAINS ISOLATED FROM PREGNANT WOMEN AT AN ACADEMIC HOSPITAL IN PRETORIA, SOUTH AFRICA
ANALYSIS OF MYCOPLASMA GENITALIUM STRAINS ISOLATED FROM PREGNANT WOMEN AT AN ACADEMIC HOSPITAL IN PRETORIA, SOUTH AFRICA Mafunise M 1, Le Roux MC 1, de Villiers BE 1, Ditsele RMM 1,2 1 Department of Microbiological
More informationPROTOCOL: OPTIMIZATION OF LENTIVIRAL TRANSDUCTION USING SPINFECTION
Last Modified: April 2018 Last Review: October 2018 PROTOCOL: OPTIMIZATION OF LENTIVIRAL TRANSDUCTION USING SPINFECTION Table of Contents 1. Brief Description 1 2. Materials and Reagents.1 3. Optimization
More informationSupplementary Information. Supplementary Figure 1
Supplementary Information Supplementary Figure 1 1 Supplementary Figure 1. Functional assay of the hcas9-2a-mcherry construct (a) Gene correction of a mutant EGFP reporter cell line mediated by hcas9 or
More informationxcelligence Real-Time Cell Analyzers
xcelligence Real-Time Cell Analyzers Application Note No. 9 A New Way to Monitor Virus-Mediated Cytopathogenicity Introduction One of the most important procedures in virology is the measurement of viral
More informationChoosing Between Lentivirus and Adeno-associated Virus For DNA Delivery
Choosing Between Lentivirus and Adeno-associated Virus For DNA Delivery Presenter: April 12, 2017 Ed Davis, Ph.D. Senior Application Scientist GeneCopoeia, Inc. Outline Introduction to GeneCopoeia Lentiviral
More information