Prevention of hepatitis C virus infection using a broad cross-neutralizing monoclonal antibody (AR4A) and epigallocatechin gallate.

The anti-hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross-genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro.

Hcmv-miR-UL22A-5p: A Biomarker in Transplantation With Broad Impact on Host Gene Expression and Potential Immunological Implications.

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.

The Pekin duck programmed death-ligand 1: cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis.

Programmed death ligand-1 (PD-L1) plays an important role in the attenuation of adaptive immune responses in higher vertebrates. Here, we describe the identification of the Pekin duck PD-L1 orthologue (duPD-L1) and its gene structure. The duPD-L1 cDNA encodes a 311-amino acid protein that has an amino acid identity of 78% and 42% with chicken and human PD-L1, respectively. Mapping of the duPD-L1 cDNA with duck genomic sequences revealed an exonic structure of its coding sequence similar to those of other vertebrates but lacked a noncoding exon 1. Homology modelling of the duPD-L1 extracellular domain was compatible with the tandem IgV-like and IgC-like IgSF domain structure of human PD-L1 (PDB ID: 3BIS). Residues known to be important for receptor binding of human PD-L1 were mostly conserved in duPD-L1 within the N-terminus and the G sheet, and partially conserved within the F sheet but not within sheets C and C'. DuPD-L1 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung and spleen and very low levels of expression in muscle, kidney and brain. Mitogen stimulation of duck peripheral blood mononuclear cells transiently increased duPD-L1 mRNA expression. Our observations demonstrate evolutionary conservation of the exonic structure of its coding sequence, the extracellular domain structure and residues implicated in receptor binding, but the role of the longer cytoplasmic tail in avian PD-L1 proteins remains to be determined.

cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis of the Pekin duck interleukin-10 receptor 1.

Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.

Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Transmembrane communication in cells chronically infected with measles virus.

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

Interferon production by lymphocytes from multiple sclerosis and non-MS patients.

Peripheral blood lymphocytes from MS patients and non-MS patients were induced to produce interferon by incubation with canine distemper, mumps, Sendai, herpes simplex I, and two strains of measles viruses. Lymphocytes from MS patients produced as much pH 2 stable interferon as did those from non-MS patients in response to induction with any of these viruses. In addition, there were no significant differences in amounts of interferon produced by lymphocytes from MS patients in three different clinical stages of the disease.

Electrophoresis of immunoglobulin G. Facilitated migration of minute amounts in agarose.

Migration of very small amounts of immunoglobulin (20 ng) is restricted in agarose electrophoresis. Incorporation of a stable protein matrix (rabbit gamma globulin 1 mg/ml) in the agarose permits unrestricted migration so that immunoelectrophoresis of this quantity of radiolabelled antibody is possible. Very small amounts of radiolabelled and non-radiolabelled antibody were subjected to successful crossed immunoelectrophoresis through barriers of antigen under conditions which provide favorable ratios of antibody to antigen. These methods should be useful for studies of antibody eluted from tissue in acquired and autoimmune diseases associated with tissue bound immunoglobulin.

Herpes simplex virus infection of the hand. Clinical features and management.

Herpes simplex virus (HSV) infection of the hand occurs predominantly in three different population groups. Young adults with a recurrent HSV II infection of the hand account for the majority of cases. A prodromal phase of up to 72 hours and a recurrence of seven to 10 days' duration occasionally associated with lymphangitis, lymphadenopathy, and lymphedema are characteristic. HSV I infection of the hand classically occurs in children with herpetic stomatitis and in health care workers infected during patient care delivery. In health care workers, the infection may last 21 to 28 days and be associated with severe pain and lymphangitis. Recurrences appear uncommon in HSV I infections. Primary infection is usually managed conservatively. Vesicle drainage for pain relief and antiviral therapy with acyclovir may be of value. For recurrent infections, acyclovir, 800 mg orally, twice daily, initiated during the prodrome in an open study of eight patients appeared effective in aborting the attack. Evaluation of long-term suppression in recurrent HSV infection of the hand is in progress.

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides.

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.

Synthesis and antiviral activity of novel acyclic nucleoside analogues of 5-(1-azido-2-haloethyl)uracils.

We present the discovery of a novel category of 5-substituted acyclic pyrimidine nucleosides as potent antiviral agents. A series of 1-[(2-hydroxyethoxy)methyl] (5-7), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] (8-10), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] (11-13) derivatives of 5-(1-azido-2-haloethyl)uracil were synthesized and evaluated for their biological activity in cell culture. 1-[4-Hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil (12) was the most effective antiviral agent in the in vitro assays against DHBV (EC(50) = 0.31-1.55 microM) and HCMV (EC(50) = 3.1 microM). None of the compounds investigated showed any detectable toxicity to several stationary and proliferating host cells.

Synthesis and antiviral and cytotoxic activity of iodohydrin and iodomethoxy derivatives of 5-vinyl-2'-deoxyuridines, 2'-fluoro-2'-deoxyuridine, and uridine.

A series of new 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine and uridine compounds (11, 16) was synthesized by the regiospecific addition of HOI to the vinyl substituent of 5-vinyl-2'-deoxyuridine (10a), 5-vinyl-2'-fluoro-2'-deoxyuridine (10b), 5-vinyluridine (10c), and (E)-5-(2-iodovinyl)-2'-deoxyuridine (4b). Treatment of the iodohydrins 11a-c with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-iodoethyl) derivatives (12a-c). In contrast, reaction of 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine (11a) with sodium carbonate in methanol afforded a mixture of 5-(1-hydroxy-2-methoxyethyl)-2'-deoxyuridine (13) and 2,3-dihydro-3-hydroxy-5-(2'-deoxy-beta-D-ribofuranosyl)- furano[2,3-d]pyrimidin-6(5H)-one (14). The most active compound, 5-(1-methoxy-2-iodoethyl)-2'-deoxyuridine (12a, ID50 = 0.1 micrograms/mL), which exhibited antiviral activity (HSV-1) 100-fold higher than that of the 5-(1-hydroxy-2-iodoethyl) analogue (11a), was less active than IVDU or acyclovir (ID50 = 0.01-0.1 micrograms/mL range). The C-5 substituent in the 2'-deoxyuridine series was a determinant of cytotoxic activity, as determined in the in vitro L1210 screen, where the relative activity order was CH(OH)CHI2 (16) greater than CH(OMe)CH2I (12a) greater than CH(OH)CH2I (11a) congruent to CH(OH)CH2OMe (13). The 2'-substituent was also a determinant of cytotoxic activity in the 5-(1-hydroxy-2-iodoethyl) (11a-c) and 5-(1-methoxy-2-iodoethyl) series of compounds, where the relative activity profile was 2'-deoxyuridine greater than 2'-fluoro-2'-deoxyuridine greater than uridine (11a greater than 11b greater than or equal to 11c; 12a greater than 12b greater than 12c). The most active cytotoxic agent (16), possessing a 5-(1-hydroxy-2,2-diiodoethyl) substituent (ED50 = 0.77 micrograms/mL), exhibited an activity approaching that of melphalan (ED50 = 0.15 micrograms/mL). All compounds tested, except for 13 and 14, exhibited high affinity (Ki = 0.035-0.22 mM range relative to deoxyuridine, Ki = 0.125) for the murine NBMPR-sensitive erythrocyte nucleoside transport system, suggesting that these iodohydrins are good permeants of cell membranes.

Synthesis of 5-[1-hydroxy(or methoxy)-2-bromo(or chloro)ethyl]-2'-deoxyuridines and related halohydrin analogues with antiviral and cytotoxic activity.

A series of new 5-(1-hydroxy-2-haloethyl)-2'-deoxyuridines (3, 6, 8) were synthesized in 60-70% yields by addition of HOX (X = Br, Cl, I) to the vinyl substituent of the respective 5-vinyl-2'-deoxyuridines (2, 5, 7). Treatment of 3a,b with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-haloethyl)-2'-(deoxyuridines (4a,b). The 5-(1-hydroxy-2-chloroethyl) (3b), 5-(1-methoxy-2-bromoethyl) (4a), 5-(1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl) (6a), and 5-(1-hydroxy-2-iodo-2-(ethoxycarbonyl)ethyl) (6b) derivatives exhibited in vitro antiviral activity (ID50 = 0.1-1 microgram/mL range) against herpes simplex virus type 1 (HSV-1). 5-(1-Hydroxy-2-bromo-2-(ethoxycarbonyl)-ethyl)-2'-deoxyuridine (6a) was the most active cytotoxic agent in the in vitro L1210 screen exhibiting an ED50 of 11 micrograms/mL relative to melphalan (ED50 = 0.15 micrograms/mL).

Efficacy of lamivudine in chronic hepatitis B patients with active viral replication and decompensated cirrhosis undergoing liver transplantation.

Liver transplantation for endstage hepatitis B virus (HBV) infection has been associated with survival inferior to that of liver transplantation in other chronic liver diseases due to HBV reinfection of the graft. Lamivudine is a new nucleoside analog with potent antiviral effects against hepatitis B. Our aim was to test its efficacy when used pre- and posttransplantation in HBV-DNA positive patients with endstage liver disease. Patients received oral lamivudine 100 mg daily both pretransplant and posttransplant. Viral serology, serum and tissue HBV-DNA and liver histology were assessed sequentially. Five consecutive patients with endstage hepatitis B were entered into the trial. Serum HBV-DNA was cleared pretransplant in all patients. Three of four transplanted patients cleared HBeAg and HBsAg postoperatively, whereas all four became negative for serum HBV-DNA (dot-blot and PCR). Liver biopsies were negative for HBV-DNA by PCR in 3 of 4 cases. Lymphocytes were negative for HBV-DNA by PCR in all cases. With follow-up of 3, 14, 16, and 26 months, two patients have normal liver enzymes and normal liver histology and two have developed recurrent hepatitis B. No significant side effects were seen. This pilot study shows that lamivudine can effectively inhibit hepatitis B virus in cirrhotic patients pretransplant and posttransplant. A lamivudine resistant mutant developed in two patients. Transplant recipients with actively replicating HBV related cirrhosis may achieve a good outcome after liver transplantation using lamivudine, but viral resistance is likely to be a significant problem.

Intracellular metabolism of 2',3'-dideoxynucleosides in duck hepatocyte primary cultures.

The intracellular fate of the potent duck hepatitis B virus (DHBV) inhibitor 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), its deamination product 2',3'-dideoxyguanosine (ddG), and the less effective DHBV-inhibitor 2',3'-dideoxycytidine (ddC) was investigated in duck hepatocyte primary cultures. After a 1-min exposure of [3H]ddDAPR to duck blood, 95% of the compound was converted to ddG. Similarly, [3H]ddDAPR was converted rapidly to ddG in duck hepatocyte primary cultures, with ddG exhibiting resistance to further catabolism. The major pathway of ddG utilization in these cells was phosphorylation, yielding a concentration of 2.1 and 1.9 microM total ddG nucleotides after 5 and 26 hr, respectively, of exposure to 4 microM ddG. Removal of exogenous ddG led to a rapid (T1/2 = 1.6 hr) decrease in the total intracellular ddG nucleotide pools. Duck hepatocytes treated with 4 microM ddC exhibited a time-dependent accumulation of ddC nucleotides, culminating in a maximum intracellular total ddC nucleotide concentration of 1.4 microM after 24-26 hr. The intracellular total ddC nucleotide level decreased with a T1/2 of 4.4 hr following the removal of exogenous ddC. The formation of ddC nucleotides was reduced in the presence of excess 2'-dideoxycytidine implicating deoxycytidine kinase in the initial step of ddC phosphorylation. A 25-fold excess of 2'-deoxycytidine had no effect on ddG phosphorylation in duck hepatocytes. However, a 92% inhibition of ddG nucleotide formation occurred in duck hepatocytes treated for 5 hr with 4 microM [3H]dG + 100 microM adenosine in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin, suggesting that, in these cells, adenosine kinase is involved in the ddG phosphorylation process.

An in vitro system for the enzymological analysis of avian hepatitis B virus replication and inhibition in core particles.

A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition.

In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses.

A group of myristic acid analogs, designed as alternative substrates for N-myristoyltransferase (NMT), were evaluated against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in vitro. Antiviral potency was increased when S or O was substituted for -CH2- in myristic acid and selectivity was affected by the presence and position of the heteroatoms and phenyl groups. A correlation was established among anti-HIV activity, Log P and Log D7.4 and between anti-HIV activity and carbonyl-heteroatom interatomic distances in the myristoyl analogs. 12-Thioethyldodecanoic acid 6 was moderately active (EC50 = 9.37 microM) against HIV-infected T4-lymphocytes (CEM-SS cell line), and it exhibited in vitro activity (EC50 = 17.8 microM) against HBV-producing 2.2.15 cell cultures derived from a human hepatoblastoma cell line (Hep G2). 12-Methoxydodecanoic acid 1 exhibited in vitro activity (EC50 = 20-30 microM) against hepatitis B in the HBV DNA-transfected 2.2.15 cell line. At a concentration of 10 microg/ml, none of the fatty acids significantly inhibited the replication of DHBV in infected hepatocytes.

A quantitative competitive PCR assay for the covalently closed circular form of the duck hepatitis B virus.

A crucial step in the establishment and maintenance of a hepadnavirus infection is the formation of a pool of covalently closed circular viral genomes in the nucleus. Changes in the size of this pool occur when an infection is established, when acute infections are resolved, and when chronic infections are treated with antiviral drugs. However, the lack of a quantitative assay for the cccDNA form of the virus has hampered study of the biology of this replication intermediate. In response to this need we have devised a sensitive and accurate competitive PCR assay that is highly selective for the cccDNA form of the duck hepatitis B virus. Since only small amounts of DNA are needed for the assay, cccDNA pool sizes can be monitored in live animals using DNA derived from needle biopsies of infected liver.

Colonic biopsy in verotoxin-induced hemorrhagic colitis and thrombotic thrombocytopenic purpura (TTP).

Sporadic cases and occasional outbreaks of hemorrhagic colitis recently have been associated with the rare Escherichia coli serotype O157:H7, which is now recognized as an important identifiable cause of bloody diarrhea in patients in whom more common gut pathogens cannot be detected. The authors report such a case in a 49-year-old woman who developed thrombotic thrombocytopenic purpura (TTP) and hemorrhagic transverse and descending colitis with a lesion having many of the features of pseudomembranous colitis. While pseudomembrane formation has been described in the hemolytic uremic syndrome (HUS), these features have not, to the authors' knowledge, been described in a patient with hemorrhagic colitis and TTP secondary to a verotoxin-producing serotype of E. coli.