Ravit Arav-Boger

Polymorphisms of the Cytomegalovirus (CMV)–Encoded Tumor Necrosis Factor–α and β-Chemokine Receptors in Congenital CMV Disease

Some congenital cytomegalovirus (CMV) infections lead to neonatal disease, whereas others have no associated sequelae. To explore a possible role for viral genes as determinants of virulence, portions of the UL144 tumor necrosis factor (TNF)–a–like receptor gene, the US28 bchemokine receptor gene, and the UL55 envelope glycoprotein B gene from 33 patients with congenital CMV infection were sequenced. Three major UL144 subtypes (A, B, and C) and 2 recombinants (A/C and A/B) were detected. Infection with the least common UL144 subtypes (A, C, A/C, and A/B) was associated with unfavorable disease outcome ( ). There was P p .04 no association between specific subtypes of the US28 and UL55 genes and outcome (P p and , respectively). Multiple genotypes (implying multiple infections) were detected .864 P p .765 in tissues from 8 of 10 autopsies. Therefore, polymorphism in the CMV-encoded TNF-a–like receptor appears to be associated with congenital CMV disease. Other CMV polymorphisms should be further evaluated for potential relevance to neonatal infection, transplantation, and acquired immunodeficiency syndrome–associated CMV diseases. Cytomegalovirus (CMV) is the most common congenital infection, affecting 1% of infants born in the United States. Why some infants have fatal or multisystem disease and others have no clinical evidence of CMV-related abnormalities, either during the neonatal period or later, is not clear [1]. The severity of fetal disease varies widely, ranging from stillbirth or neonatal death due to multisystem disease to no abnormalities. Similarly, the outcome of congenital CMV infection ranges from no apparent impairments to significant central nervous system (CNS) damage, manifested as mental retardation, cerebral palsy, hearing loss, and impaired vision, appearing alone or in combination. Although clinical and epidemiological factors associated with a greater risk of CNS damage have been identified [2, 3], the role of putative viral virulence genes as determinants of the outcome of congenital CMV infection has not been explored. A substantial portion of the human CMV genome encodes proteins with the potential to affect virulence through immune evasion, molecular mimicry, or interference with host chemokines

Cytomegalovirus (CMV)–Encoded UL144 (Truncated Tumor Necrosis Factor Receptor) and Outcome of Congenital CMV Infection

BACKGROUND Cytomegalovirus (CMV) is the most common congenital infection in humans. The effect of viral strains on the outcome of congenital CMV is debated. We evaluated whether UL144 polymorphisms in amniotic fluid from CMV-infected Italian women were associated with terminations of pregnancy, subsequent disease in their offspring, or viral load. METHODS The study was nested within a prenatal CMV program. We sequenced the UL144 gene from 66 amniotic-fluid samples, without knowledge of pregnancy outcome. We performed data analyses on 56 samples for which all information was available. RESULTS Genotype C was associated with termination of pregnancy (P=.03). Genotype B was associated with fewer terminations of pregnancy (P=.003). A possible association was found between genotype C and symptomatic disease in newborns (odds ratio, 8.81 [95% confidence interval, 0.48-164.02]; P=.05). There was no association between specific genotype and the viral load in amniotic fluid. Symptomatic newborns who had the most common UL144 genotype (B) were more likely to have higher viral loads than were asymptomatic infants (P=.003). CONCLUSIONS UL144 polymorphisms may be associated with the outcome of congenital CMV infection. Larger studies should be conducted to confirm this association, before genotype analysis can be used, along with other factors, in considering terminations of pregnancy.

Portal vein thrombosis caused by protein C and protein S deficiency associated with cytomegalovirus infection

A 4-month-old girl who was examined because of splenomegaly had portal vein thrombosis, which apparently resulted from a combination of cytomegalovirus infection and deficiency of both protein S and protein C. The cytomegalovirus infection, by damaging endothelial cells, may have triggered a cascade of events that was ultimately expressed as portal vein thrombosis resulting from deficiency of proteins S and C.

Human Cytomegalovirus–Encoded α-Chemokines Exhibit High Sequence Variability in Congenitally Infected Newborns

Most congenital human cytomegalovirus (HCMV) infections are asymptomatic, but some lead to severe disease. We hypothesized that differences in disease manifestations may be partially explained by differences in viral strains. We recently reported an association between unique long (UL) 144 gene polymorphisms and clinical disease. We now report on the sequence heterogeneity of 2 potential HCMV virulence genes that encode alpha -chemokines: UL146 and UL147. These 2 genes were highly divergent in cultured isolates obtained from 23 newborns with congenital HCMV infection and were difficult to categorize. Unlike our findings for the contiguous UL144 gene, no specific UL146 or UL147 genotype was associated with disease outcome.

Clearance of adenoviral hepatitis with ribavirin therapy in a pediatric liver transplant recipient

Adenovirus (AdV) infections, localized or disseminated, are being increasingly recognized in immunocompromised patients including bone marrow and solid organ transplant recipients and HIV-1-infected patients. AdV infection in pediatric liver transplant recipients may present as hepatitis in the transplanted liver and is usually diagnosed by immunohistochemical staining of liver tissue. Recommended therapies have included withdrawal of immunosuppressive agents and in some cases intravenous ribavirin. There has been no previous report of successful clearance of AdV hepatitis with ribavirin in a pediatric liver transplant recipient. We report the clearance of AdV hepatitis in a child who received two liver transplants, developed AdV hepatitis and was successfully treated with intravenous ribavirin. Diagnosis and monitoring of her AdV infection were enhanced through the use of a newly described polymerase chain reaction for AdV.

Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers

Background Artesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers. Methods Four artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity. Results Artemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC50 for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein. Conclusions Artemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.

An Artemisinin-Derived Dimer Has Highly Potent Anti-Cytomegalovirus (CMV) and Anti-Cancer Activities

We recently reported that two artemisinin-derived dimers (dimer primary alcohol 606 and dimer sulfone 4-carbamate 832-4) are significantly more potent in inhibiting human cytomegalovirus (CMV) replication than artemisinin-derived monomers. In our continued evaluation of the activities of artemisinins in CMV inhibition, twelve artemisinin-derived dimers and five artemisinin-derived monomers were used. Dimers as a group were found to be potent inhibitors of CMV replication. Comparison of CMV inhibition and the slope parameter of dimers and monomers suggest that dimers are distinct in their anti-CMV activities. A deoxy dimer (574), lacking the endoperoxide bridge, did not have any effect on CMV replication, suggesting a role for the endoperoxide bridge in CMV inhibition. Differences in anti-CMV activity were observed among three structural analogs of dimer sulfone 4-carbamate 832-4 indicating that the exact placement and oxidation state of the sulfur atom may contribute to its anti-CMV activity. Of all tested dimers, artemisinin-derived diphenyl phosphate dimer 838 proved to be the most potent inhibitor of CMV replication, with a selectivity index of approximately 1500, compared to our previously reported dimer sulfone 4-carbamate 832-4 with a selectivity index of about 900. Diphenyl phosphate dimer 838 was highly active against a Ganciclovir-resistant CMV strain and was also the most active dimer in inhibition of cancer cell growth. Thus, diphenyl phosphate dimer 838 may represent a lead for development of a highly potent and safe anti-CMV compound.

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Recombinant luciferase-expressing human cytomegalovirus (CMV) for evaluation of CMV inhibitors

Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.

Loss of Linkage Disequilibrium and Accelerated Protein Divergence in Duplicated Cytomegalovirus Chemokine Genes

Human CMV (hCMV) encodes several captured chemokine ligand and chemokine receptor genes that may play a role in immune evasion. The adjacent viral alpha-chemokine genes UL146 and UL147 appear to have duplicated subsequent to a recent gene capture event. Sequence data from multiple hCMV isolates suggest accelerated protein evolution in one of the paralogues, UL146. Extensive sequence variation was noted throughout the more rapidly evolving paralogue, although significant variation was also observed within the more slowly evolving gene, especially within a region corresponding to a possible signal peptide. In contrast to the haplotype structure observed for other hCMV genes, the distribution of nucleotide variants indicates a marked loss of linkage disequilibrium within UL146 and to a lesser extent UL147. Despite evidence of accelerated protein evolution, the rate of nonsynonymous to synonymous substitutions (dN/dS) in the more rapidly evolving paralogue was not indicative of neutral evolution, but of moderate purifying selection. The data presented here provides a unique opportunity to study the mechanisms by which a recently duplicated pair of genes has diverged and suggests a role for recombination.