An acyclic phosphonate prodrug of HPMPC is effective against VZV in skin organ culture and mice.

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 µM, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.

Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Macrolide-Resistant Mycoplasma pneumoniae in the United States as Determined from a National Surveillance Program.

There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 µg/ml, whereas MICs for MRMp were 16 to 32 µg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence.

Brincidofovir treatment of acyclovir-resistant disseminated varicella zoster virus infection in an immunocompromised host.

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet.

Helicase-primase as a target of new therapies for herpes simplex virus infections.

The seminal discovery of acyclovir 40 years ago heralded the modern era of truly selective antiviral therapies and this drug remains the therapy of choice for herpes simplex virus infections. Yet by modern standards, its antiviral activity is modest and new drugs against novel molecular targets such as the helicase-primase have the potential to improve clinical outcome, particularly in high-risk patients. A brief synopsis of current therapies for these infections and clinical need is provided to help provide an initial perspective. The function of the helicase-primase complex is then summarized and the development of new inhibitors of the helicase-primase complex, such as pritelivir and amenamevir, is discussed. We review their mechanism of action, propensity for drug resistance, and pharmacokinetic characteristics and discuss their potential to advance current therapeutic options. Strategies that include combinations of these inhibitors with acyclovir are also considered, as they will likely maximize clinical efficacy.

The genetic basis of human cytomegalovirus resistance and current trends in antiviral resistance analysis.

Infections due to resistant human cytomegalovirus (CMV) are an emerging problem, particularly in immunocompromised hosts. When managing such patients, clinicians should be aware of the possibility of developing CMV antiviral resistance, especially while on prolonged therapy or if severe immunosuppression is present. CMV resistance to current antiviral agents is mediated by alterations in either the UL97 kinase or DNA polymerase, encoded by the UL97 and UL54 genes, respectively. UL97 mutations are capable of conferring resistance to ganciclovir, while UL54 mutations can impart resistance to ganciclovir, cidofovir, and foscarnet. If treatment failure is suspected to be due to antiviral resistance, CMV resistance analysis should be obtained. Phenotypic resistance assays performed on clinical isolates measure antiviral susceptibilities directly, but are laborious and time-consuming. Therefore, genotypic resistance analysis has become the more common means of diagnosing CMV resistance. Mutations in UL97 or UL54 may be clinically associated with resistance, but their effect on antiviral susceptibility must be confirmed by marker transfer techniques such as recombinant phenotyping.

Carpal and fetlock conformation of the juvenile Thoroughbred from birth to yearling auction age.

REASONS FOR PERFORMING STUDY: There is little information available about conformational changes in the forelimbs of growing foals. OBJECTIVES: To describe the conformation of the carpus and fetlock of Thoroughbred foals from birth to yearling sale age. METHODS: Subjective assessments of the fetlock and carpal conformation of 119 Thoroughbred foals were made within the first month of life and then at 30 day intervals until at least age 120 days. At least 70 subjects were examined further at 60 day intervals until September of their second year. Conformation grades are reported for 5 age groups: first 7 days and at a mean of 46, 176, 362 and 525 days. The conformation of all available sires and dams of subjects was also graded. RESULTS: All subjects demonstrated carpal deviations, such as valgus, outward rotation and offset, and approximately 30% had fetlock deviations. Heavier birth weights were associated with carpal offset and fetlock inward conformation at most ages, and heavier yearlings were more likely to be carpal valgus. The carpal conformation of the sire (offset and outward rotation) was associated with similar yearling carpal conformation. During the study period, the carpal conformation of Thoroughbred foals became less valgus and more offset. Fetlock conformation became more inwardly deviated during the first 6 months of the study. CONCLUSIONS: Carpal and fetlock conformation change greatly in Thoroughbred foals up to age 18 months. The phenotype of the sire can be associated with yearling carpal conformation and bodyweight, particularly at birth and as yearlings, is associated with yearling fetlock and carpal conformation. POTENTIAL RELEVANCE: New factors associated with forelimb conformational deviations have been identified that may help breeders better to manage young racing stock.

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Bifunctional protein conferring enhanced green fluorescence and puromycin resistance.

A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a selectable/screenable marker was demonstrated by the ease with which a recombinant guinea pig cytomegalovirus containing EGFP-puro was isolated by a combination of puromycin selection and screening for green fluorescence. We conclude that EGFP-puro is a compact and versatile marker that should prove useful for recombinant virus and transgenic cell line construction, particularly in applications in which coding capacity is limited.

Requirement for uracil-DNA glycosylase during the transition to late-phase cytomegalovirus DNA replication.

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.

A review of genetic differences between limited and extensively passaged human cytomegalovirus strains.

The complete genetic content of human cytomegalovirus (HCMV) has been difficult to determine, since most strains studied in the laboratory have been extensively passaged in human fibroblast cultures which can change the genetic content as well as the biological properties of the virus. Approximately 13 kb of novel DNA sequences located near the right edge of the unique long (UL) component of the genome has been discovered in Toledo, clinical isolates and certain stocks of Towne. This region of novel sequence, designated the UL/b' region, encodes several interesting proteins including vCXC-1, a potent IL-8 homologue, and UL144, a member of the TNF receptor family. This region is missing from the prototypic laboratory variants of Towne and AD169. In contrast to Toledo and other low passage isolates which have relatively small repeats bracketing the UL component, the Towne and AD169 laboratory variants contain large (>10 kb) b/b' repeats. The large size of these repeats in AD169 and Towne appear to have arisen as compensation for the loss of sequences from the UL/b' region that existed in less passaged variants of these strains. Consequently, many of the haploid genes at the left edge of the prototypic wild-type (wt) UL component are diploid in AD169 and Towne. We hypothesise that this plasticity of the genome at the right edge of the UL component results from extensive passage and adaptation to replication in fibroblasts in vitro. Further work will be required to understand the complete genetic content of wt HCMV.

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation.

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

The impact of margin of resection on outcome in pediatric nonrhabdomyosarcoma soft tissue sarcoma.

BACKGROUND/PURPOSE: Because the management of pediatric nonrhabdomyosarcoma soft tissue sarcomas (NRSTS) is determined by extrapolation from adult studies, the effect of margin of tumor resection and postoperative radiation therapy (RT) on local tumor recurrence in children has not been assessed. METHODS: Records of NRSTS patients from a single institution were reviewed with regard to demographic data, TNM staging, grade, histological type and site of primary tumor, RT, and local tumor recurrence. The margin of resection was determined by pathological review and did not necessarily reflect operative margins. RESULTS: Eighty-eight clinical group I patients were treated over a 30-year period. The most common histological tumor subtypes were synovial cell sarcoma (n = 26), malignant fibrous histiocytoma (n = 17), and fibrosarcoma (n = 7). The mean age was 9.4 years (range, 0 to 29 years). Thirty-four patients had high-grade tumors. Two of ten patients with low-grade tumors and margins less than 1 cm, including one of five who had received RT, had a local recurrence. Patients with low-grade tumors and margins greater than 1 cm (n = 44) had a lower recurrence rate (2 of 44, 4.5%). None of these patients had received RT. Fourteen patients with high-grade tumors had margins less than 1 cm. Seven of these had RT and had no recurrence. Three of the seven patients who received no RT had a recurrence (42.9%). None of the 20 patients with high-grade tumors and margins greater than 1 cm received RT; four of these patients had recurrences (20%). Seven of the 12 irradiated patients (58.3%) had serious radiation-associated complications (wound dehiscence, fracture, growth retardation, and joint dysfunction). CONCLUSIONS: Grade alone does not determine the rate of local recurrence. In both low- and high-grade tumors, a pathological margin of resection greater than 1 cm reduced local recurrence. Radiotherapy provided no advantage in low grade tumors but did decrease local recurrence rates in high-grade tumors with less than 1 cm pathological margins.

A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency.

Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCDelta97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, the UL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.

Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.

Anal leiomyoma in a Holstein heifer.

A 2-year-old heifer was presented with masses on her anus that were interfering with cervical manipulation during embryo flushing. The masses had broad stalks attached within the anal sphinchter. Recovery was without incident after surgical resection. No recurrence of the masses had occurred 3 months later. Histologic diagnosis was benign leiomyoma.

Reassessing the organization of the UL42-UL43 region of the human cytomegalovirus strain AD169 genome.

A polymorphism in the UL42-UL43 region of the human cytomegalovirus genome has been characterized by nucleotide sequence analysis, revealing a 929-bp insertion following nt 54,612 relative to the published strain AD169-UK genome sequence (M.S. Chee et al., 1990, Curr. Top. Microbiol Immunol. 154, 125-170). Although AD169-UK exhibited polymorphism in this genomic region, other CMV strains (Towne, Toledo, and AD169-ATCC) carried only the newly characterized longer form. The additional sequence altered the assignment of UL42 and UL43 open reading frames. UL42 decreased in size from 157 to 125 codons, retaining 76 of the previously reported carboxyl terminal codons, and UL43 increased in size from 187 to 423 codons, retaining 185 of the previously reported amino terminal codons. This additional sequence makes UL43 a more conserved betaherpesvirus US22 family member. Only AD169-UK exhibited restriction fragment length polymorphism in this region, suggesting that a deletion occurred during the propagation of this strain in cell culture. The additional sequence should be considered a bona fide part of the cytomegalovirus genome and the AD169 genome size should be corrected to 230,283 bp.

Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication.

Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.

Modeling combinations of antiretroviral agents in vitro with integration of pharmacokinetics: guidance in regimen choice for clinical trial evaluation.

We propose a method for the selection of doses and dosing schedule for drugs to be used in combination. This approach uses the simulation of steady-state concentrations of the drugs in the combination and overlays these concentrations onto a three-dimensional effect surface. The MacSynergy II program is used to construct the three-dimensional drug interaction surface from the direct evaluation of drug combination effect in vitro. The study examined the combination of an inhibitor of the human immunodeficiency virus protease, A-77003, and the nucleoside analog zidovudine. Zidovudine concentrations from a steady-state interval were simulated on the basis of the administration of 100 mg every 12 h by mouth, while for A-77003 simulation profiles were for intravenous administration of 800 mg every 4 h as well as a continuous infusion of 200 mg/h. The average percentage of the maximal effect was taken as a measure of regimen effectiveness. Three different schedules of administration were examined. If both drugs were to be administered simultaneously, the model predicts a mean maximal effect of a steady-state interval (12 h) of 67%. If the drug doses were offset by 2 h, the mean maximal effect predicted was 71%. If A-77003 was to be given by continuous infusion, the mean maximal effect predicted was 90%. This method holds promise as a way of quickly evaluating potential combinations of agents that takes into account the drug interaction in a mathematically robust way and that allows the evaluation of the effect of each drug's pharmacokinetic profile.