Sofosbuvir and simeprevir combination therapy in the setting of liver transplantation and hemodialysis.

We report safety, tolerability, and 12-week sustained virologic response with half-standard dose sofosbuvir and standard-dose simeprevir combination therapy in a hepatitis C virus genotype 1a-infected liver transplant recipient on hemodialysis - uncharted territory for sofosbuvir-based therapy. The patient was a non-responder to prior treatment with pegylated interferon plus ribavirin. Sofosbuvir efficacy was maintained despite pill-splitting and administration of half-standard dose, 200 mg per day. No drug-drug interactions were noted with tacrolimus-based immunosuppression. Laboratory tests remained stable or improved during therapy. Our observation, if reproduced in a larger study, may lead to significant improvement in clinical outcomes and cost savings in this patient population.

Prenylation of HDAg and antiviral drug development.

Hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease. Current medical therapies are unable to effectively eradicate HDV infections. Research into the molecular virology of the HDV life cycle has revealed a fascinating collection of biology. These insights are now beginning to be translated into new potential treatment strategies. For example, an essential step in the virus assembly process involves the post-translational lipid modification of a specific HDV protein, namely prenylation of large delta antigen. Preventing prenylation abolishes virus particle formation. Drugs capable of specifically inhibiting prenylation have been developed for use in humans. These agents represent a new class of antiviral agents, with HDV as a first target. Here, a brief review of the HDV life cycle emphasizing the role of prenylation is presented along with implications for drug development and therapy.

Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

Use of a prenylation inhibitor as a novel antiviral agent.

No specific therapy exists for hepatitis delta virus (HDV), which can cause severe liver disease. Molecular genetic studies have implicated the prenylation site of large delta antigen as a critical determinant of HDV particle assembly. We have established a cell culture model which produces HDV-like particles, and we show that delta antigen prenylation can be pharmacologically inhibited by the prenylation inhibitor BZA-5B. Furthermore, BZA-5B specifically abolishes particle production in a dose-dependent manner. These results demonstrate that the use of such a prenylation inhibitor-based antiviral therapy may be feasible and identify a novel class of potential antiviral agents.

Cervical and thoracic vertebral malformation ("weak neck") in Colombia lambs.

The purpose of this study is to describe a developmental defect of the caudal cervical and cranial thoracic vertebrae in 11 purebred Colombia lambs. The lambs were either affected at birth, or developed the condition within the first 18 days of age. Cervicothoracic kyphosis, with a compensatory cervical lordosis and ataxia were common; 8 lambs had abnormal head posture, characterized by inability to lift the head from the ground. One lamb had rigid head and neck, and had to move the entire body to look to the left or right. Neurological signs included ataxia, tetraparesis, diminished conscious proprioception, and increased patellar and triceps reflexes. One lamb had inspiratory stridor because of compression of the trachea in the area overlying the abnormal vertebrae (cervical vertebrae 6 [C6] and 7 [C7]). Radiographic and pathological abnormalities included malalignment and malarticulation of the caudal cervical and cranial thoracic spine, rounded cranioventral margins in the bodies of vertebrae C7 and T1, wedging of the intervertebral disc spaces between C6 and T1 vertebrae, and hypoplasia of the dens. Pathological changes in the soft tissues included hypoplasia of the cervical epaxial and hypaxial musculature, with associated focal areas of myodegeneration. Mild Wallerian axonal degeneration, compatible with a mild cord compression syndrome, was found in 3 lambs in the cervicothoracic spinal cord adjacent to the vertebral anomalies. The concentrations of copper and selenium in blood, plasma, or tissues were normal in 10 of 11 lambs. All but one of the lambs in which pedigree information was provided were genetically related. Siblings born as twins to 5 of the affected lambs were normal, but both lambs from one twin pregnancy were affected. Owners reported that breeding stock had been shared among the ranches. Because of the close familial relationships of the affected lambs, the condition is suspected to have a hereditary basis.

Colostral transfer of Bacteroides nodosus antibodies in sheep.

Ovine contagious foot rot may cause lameness in sheep, resulting in decreased wool growth and low weight gain. Affected neonatal lambs are difficult to treat, and treatment is labor intensive; thus, a method of prevention is warranted. Vaccination of ewes with a multivalent vaccine in an oil adjuvant induced development of antibody to the somatic O antigen of Bacteroides nodosus, and this antibody was detected in serum of newborn lambs after consumption of colostrum from the vaccinated ewes. Antibody titers were determined in 48 unvaccinated ewe/lamb pairs, and in 50 once-vaccinated and 78 twice-vaccinated pairs. Serum and colostrum O-agglutinin titers to B nodosus were determined by a microtitration agglutination test. Lambs from vaccinated ewes had significantly (P less than 0.05) higher O-agglutinin titers than those from unvaccinated ewes, and double vaccination of ewes resulted in the highest potentially protective titers (greater than 1:2,400) in ewes and lambs.

Identification of a prenylation site in delta virus large antigen.

During replication, hepatitis delta virus (HDV) switches from production of small to large delta antigen. Both antigen isoforms have an HDV genome binding domain and are packaged into hepatitis B virus (HBV)-derived envelopes but differ at their carboxy termini. The large antigen was shown to contain a terminal CXXX box and undergo prenylation. The large, but not the small, antigen formed secreted particles when expressed singly with HBV surface antigen. Mutation of Cys211 in the CXXX box of the large antigen abolished both prenylation and particle formation, suggesting that this site is important for virion morphogenesis.

trans-dominant inhibition of human hepatitis delta virus genome replication.

Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the delta antigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to be required for genome replication. Another genome type (termed the L genome) extends the reading frame by 19 amino acids as a result of a single base change. Replication of the S and L genomes was studied in cultured fibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in a codelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was also observed following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutational analysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame's extra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.

In vitro-synthesized hepatitis delta virus RNA initiates genome replication in cultured cells.

Monomers of the genomic strand of hepatitis delta virus RNA were transcribed in vitro and then delivered to NIH 3T3 fibroblasts by using a liposome fusion technique. After 7 days, genome replication was detected, but only in fibroblasts that stably expressed the delta antigen. Sequence analysis of the replicated products identified them as faithful copies of the hepatitis delta virus genome found in virions.

Randomized efficacy trials of long-acting oxytetracycline in neonatal pigs.

Prophylactic efficacy of 100 mg of long-acting oxytetracycline (OTC) given IM to neonatal pigs within 12 hours of birth was evaluated in a swine herd. The herd had a history of increased neonatal mortality, diarrhea, foot abscess, and arthritis in nursing pigs. Two trials were conducted in which liters and individual pigs were the treatment groups of interest. In both trials, OTC treatment failed to reduce mortality, diarrhea, or arthritis or the need for subsequent antimicrobial therapy (P greater than 0.05). Preweaning weight gains were not increased (P greater than 0.05) in treated pigs. However, in the individual pig trial, foot abscess rates were significantly (P = 0.01) lower in treated pigs (3.7%) than in nontreated pigs (8%). Aerobic bacteria isolated from pigs with diarrhea, arthritis, or foot abscess had minimum inhibitory concentrations for OTC greater than or equal to 64 micrograms/ml or were classed as resistant on the basis of disk-diffusion tests.

Economics of Brucella ovis control in sheep: epidemiologic simulation model.

The epidemiology and economics of Brucella ovis control in a hypothetical, commercial sheep flock (100 rams and 2,500 ewes) were investigated. The investigation consisted of an epidemiologic simulation model, reported here, and a decision-tree analysis, reported in a companion paper. The epidemiologic model was designed to simulate the transmission and persistence of B ovis in a ram flock during the mating season as well as the nonmating (isolation) season. A constant contact rate was selected for the nonmating season and a varying contact rate was selected for the mating season to reflect changes in numbers of ewes in estrus. These contact rates were used to evaluate all possible combinations of 5 control alternatives for flock infection rates ranging from 0% to 38%. Vaccination was found to be more effective as a control strategy when the prevalence of flock infection was high (greater than 15%); however, it did not substantially reduce B ovis transmission when the prevalence of flock infection was low (less than 10%). The effect of increasing vaccine efficacy from 40% to 80% had minimal effect on incidence of new cases. The speed with which B ovis could be eradicated depended on the initial prevalence of infection and the screening tests used (palpation, semen testing for leukocytes, and ELISA). All combinations of screening tests verified the usefulness of palpation. Simulation model results indicated that it may be feasible to eradicate B ovis from flocks with moderate to high (10% to 38%) prevalence of infection by culling on the basis of 2 sequential tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Economics of Brucella ovis control in sheep: computerized decision-tree analysis.

The epidemiology and economics of Brucella ovis control in a hypothetical, commercial sheep flock (100 rams and 2,500 ewes) were investigated. The investigation consisted of an epidemiologic simulation model, reported in a companion paper, and a decision-tree analysis, reported here. It was predicted from the simulation model that B ovis could be eradicated successfully in 2 test periods (less than 1 year) from a flock by using intensive screening and culling. A computerized decision-tree program was used to determine the economically optimal control strategy among several alternatives. Two versions of the program were used to determine the optimal alternative, based on minimizing the expected monetary loss (deterministic) and minimizing the associated risk (stochastic). The economically optimal alternative was to screen the rams by means of palpation, semen testing, and ELISA prior to the mating season. Rams positive to any test were culled. After the mating season was completed, the optimal action was to use ELISA for the remaining rams and to cull all that were ELISA positive. The cost of this alternative was approximately $6,150, or less than one half the annual cost of a vaccination program ($12,800) or no program ($13,550). Continuing palpation and semen testing were considered worthwhile on the basis of detecting new cases of B ovis infection and in maintaining high flock fertility. Similarly, the cost of annual use of ELISA was small (approximately $100), compared with the potential cost of not detecting a new case of B ovis infection.

Isolation of sigma-28-specific promoters from Bacillus subtilis DNA.

Sigma-28-RNA polymerase is a minor form of RNA polymerase found in vegetative cells of Bacillus subtilis which utilizes promoter sites distinct from those recognized by the major RNA polymerase. We have isolated a collection of cloned B. subtilis DNA segments that contain in vitro promoter sites for sigma 28-RNA polymerase by screening a bacteriophage lambda library of B. subtilis genomic fragments. At least nine new sigma 28-specific promoter sites have been identified in this collection, and four have been partially mapped for further study. Our strategy employed a mix of RNA probes prepared by in vitro transcription with sigma 28-RNA polymerase of total B. subtilis DNA EcoRI and HindIII fragments. Over 70% of the unique clones identified contain sigma 28-specific promoter sites, suggesting that the method may have general application for identification of promoter-containing sequences. The efficiency with which sigma 28-specific promoters are detected is consistent with there being a relatively small number of such sites in the B. subtilis genome of which twelve have been cloned.

Anthelmintic treatment of nonpastured dairy cows in California.

Many dairy cattle are housed under drylot conditions, which generally are not considered conducive to elevated populations of gastrointestinal parasites. Thus, an examination of the potential benefits to be obtained by anthelmintic treatment of lactating cows under these circumstances was initiated. Because there were no statistically significant differences in milk production between control and treated groups, routine deworming cannot be considered economically sound under the management conditions described.