Current Research | Seattle Children’s Hospital

Current Research | Seattle Children’s Hospital

There are several kinds of herpesviruses that can cause blisters and pain in various parts of the body. Usually, herpes simplex 2 (HSV2) causes lesions in the genital area, resulting in genital herpes. Pediatr Infect Dis J. This review focuses on the spectrum of cerebral manifestations caused by these viruses, particularly on non-congenital presentations. Corey Casper from Fred Hutchinson Cancer Research Center – played key roles in the discovery and characterization of KSHV and related viruses in non-human primates, and in the role of the oral environment in the acquisition and transmission of the virus. The HHV6 variant A was detected in 2 patients by sequence analysis, and HHV6 protein was detected by immunomicroscopy in a patient who underwent biopsy secondary to progressive clinical and neuroradiographic findings. Barkeloo, are attending the 2014 BIO International Convention, 23-26 June 2014 at the San Diego Convention Center, San Diego, CA.

lower educational level, residency outside greater Athens), whereas HSV-2 was associated with sexual behavioural factors (e.g. Thus, the relative inefficiency of experimental upper respiratory tract infection was attributable to limited liquid retention in this site. JAT is funded by the NIH (grant numbers HL67155 and HL65652), the Abby Glaser Fund, the Vivian L Smith Foundation, and the Children’s Cardiomyopathy Foundation. The alliance’s mandate is to facilitate research, training and clinical care in infection-related cancers, and to provide fiscal and administrative management to collaborative programs. The Alliance is based at the Uganda Cancer Institute in Kampala, where the epidemiology, pathophysiology, natural history and treatment of infection-related cancers has been studied since 2004. There is no cure for HSV1, but treatments are available that can shorten the length of an outbreak and make cold sores less painful. Once someone has been infected with the virus, it stays in the body.

Pediatrics. More recently, Rose obtained an administrative supplement to the program project enabling a collaboration with Drs. Damien Chaussabel and Peter Linsley of the Benaroya Research Institute to conduct a genome-wide comprehensive analysis of global KSHV, cellular and immune gene expression in their complement of unique tissue samples from Uganda. TRecent studies have highlighted the importance of the oral environment for the biological life cycle and transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), and its association with the AIDS-related malignancy Kaposi’s sarcoma (KS). Ranked in all 10 specialties on U.S.News & World Report’s 2013-14 “America’s Best Children’s Hospitals” list and among the Top 10 on Parents magazine’s 2013 “Best Children’s Hospitals” list, Nationwide Children’s Hospital is one of the nation’s largest not-for-profit freestanding pediatric healthcare networks providing care for infants, children and adolescents as well as adult patients with congenital disease. Additional studies suggest that the oropharynx is an important site for the initial acquisition of a KSHV infection. Little is known, however, regarding the biology of KSHV in oral keratinocytes at any of the levels of virus recognition by epithelial cell receptors; biological response to infection of epithelial cells; or production and characterization of infectious virions from epithelial cells.

The Rose lab’s is working to identify and characterize the viral and cellular factors involved in KSHV entry and infection of oral keratinocytes. In 1999, Rose, in association with collaborators at the University of Washington, sequenced the KSHV glycoprotein B, a major constituent of the KSHV virion envelope. They identified an “RGD” motif in glycoprotein B which is known to bind to members of the integrin family of cell surface receptors, including integrin alphaV/beta3. Rose and colleagues obtained a U.S. patent for the use of glycoprotein B peptides, polynucleotides and antibodies for the diagnosis of KSHV infection and eliciting immune responses in KSHV vaccines. Dr. Jacques Garrigues, a longtime member of the Rose lab team, has continued the work on the role of KSHV glycoprotein B in KSHV entry.

Garrigues determined that a peptide containing the KSHV glycoprotein B RGD motif specifically binds to integrin alphaV/beta 3 and function blocking antibodies inhibit adhesion of cells to the RGD peptide. Using affinity-purified integrins and confocal microscopy, he demonstrated that alphaV/beta3 integrin bound to the glycoprotein B RGD motif and to KSHV virions, demonstrating direct receptor-ligand interactions. Specific alphaV/beta3 antagonists, including cyclic and dicyclic RGD peptides and alphaV/beta3 function-blocking antibodies, strongly inhibited KSHV infection. In 1997, Rose and colleagues at the University of Washington discovered a new herpesvirus, retroperitoneal fibromatosis herpesvirus (RFHV), in a macaque tumor that is similar to Kaposi’s sarcoma. The Rose lab has characterized RFHV by sequencing critical regions of the viral genome, including the divergent locus B; ORF73 – the latency-associated nuclear antigen (LANA); and ORF59 – the DNA polymerase processivity factor. More recently, the complete genome sequence of RFHVMn, from a pig-tailed macaque (M. nemestrina), was determined by next-gen sequencing of DNA from an RF tumor.

Current Research | Seattle Children’s Hospital
The sequence was obtained from an archived frozen RF tumor carrying ~3 RFHV genomes per cell by Illumina-based sequencing of the complete complement of DNA present in the tumor, reducing the DNA reads from the macaque genome bioinformatically and performing a de novo assembly of the RFHV genome sequence. Comparison of the RFHVMn and KSHV genomes revealed close genetic and genomic similarities, suggesting a common biology, life cycle and pathology. These similarities demonstrate that RFHV infection in macaques is a close animal model of KSHV infection in humans. In 2000, Rose and others determined that two homologs of KSHV existed in macaques and several other non-human primate species, constituting two distinct rhadinovirus lineages in Old World primates. KSHV and RFHV grouped within the RV1 rhadinovirus lineage with other closely related homologs of KSHV. The second RV2 rhadinovirus lineage consisted of rhesus rhadinovirus (RRV), pig-tailed rhadinovirus (MneRV2) and other closely related rhadinoviruses. Non-human primates are infected with viruses from both lineages.

An RV2 rhadinovirus infecting humans has not yet been discovered. The Rose lab has shown that the macaque RV1 rhadinoviruses are present in the spindeloid tumor cells of the macaque retroperitoneal fibromatosis tumors, while the macaque RV2 rhadinoviruses are present in the lymphoid tumor cells of B-cell and T-cell lymphomas of macaques with SIV-associated AIDS. The Rose lab is characterizing the macaque RV1 (RFHVMn and RFHVMm) and RV2 (RRV and MneRV2) rhadinoviruses from rhesus (Mm) and pig-tailed macaques (Mn) to better understand their biology and differences in virus life cycle, transmission and pathology. The goal is to use sequence and functional comparisons with KSHV to determine the roles of critical viral genes to better understand KSHV and its role in the development of KS. The Rose lab cloned and sequenced the ORF 73 LANA homologs of the pig-tailed macaque RV1 and RV2 rhadinoviruses, RFHVMn and MneRV2, since pig-tailed macaques are the major macaque species in the WaNPRC. Several differences were observed between the RV1 and RV2 LANA homologs, including a large internal repeat region that was present in the LANAs of both HHV8 and RFHV but absent in the RV2 LANA. The repeat regions of both HHV8 and RFHV were characterized by repetitive reiterations of glutamic acid residues.

The RRV and MneRV2 LANA homologs were significantly shorter and lacked the repeat region. A comparison of the RFHV and HHV8 LANA sequences showed a low but significant sequence conservation throughout the N and C terminal regions (~30%) and the presence of conserved chromosome binding and nuclear localization signals. We determined that the monoclonal LN53 anti-KSHV LANA antibody reacted with the RFHVMn LANA. Using this antibody we showed that the spindeloid tumor cells in the macaque retroperitoneal fibromatosis tumor expressed LANA which correlated with the quantitative PCR data showing that the tumors contained high levels of RFHV DNA (~2-4 viral genomes/cell). This data strongly supported an etiological association of RFHV with the retroperitoneal fibromatosis tumors. Analysis of the nuclear localization signal of KSHV LANA revealed a strong sequence and functional conservation with RFHV LANA. We determined that the nuclear localization signal was bifunctional, allowing the LANA proteins to be imported into the nucleus using both the classical and non-classical nuclear import pathways.

The multifunctional nuclear localization signal may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency. We have cloned and expressed the ORF 59 homologs of the RV1 and RV2 rhadinoviruses from the chimpanzee and three species of macaques. We found that the ORF59 homologs of the RV1 and RV2 rhadinoviruses are highly conserved with distinct phylogenetic clustering within the two rhadinovirus lineages. A rabbit antiserum was developed against a conserved sequence in the macaque RV2 ORF 59 sequences. This antiserum was used to show the presence of ORF59 positive infected cells within the differentiating layer of the epidermis. This strongly supports the idea that the differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses. We have analyzed the promoters for the RTA homologs in the rhesus rhadinovirus (RRV) and Kaposi’s sarcoma-associated herpesvirus (KSHV).

We identified cells that are not permissive for RRV replication and recapitulate the latent KSHV infection and reactivation processes. We identified a critical Sp1 element in the RRV RTA promoter that was conserved in the KSHV RTA promoter. These studies showed that while the outcome of KSHV infection was determined by LANA inhibition of the RTA promoter activity, the outcome of RRV infection was determined by host factors, such as Sp1. The Rose lab developed a novel technique using consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the identification of distantly related genes. We have developed an interactive software program and website for the design of CODEHOP PCR primers for the identification of distantly related genes (iCODEHOP). We are working closely with the Washington National Primate Research Center to identify new pathogens infecting primates maintained at the center, and are developing CODEHOP PCR assays to detect novel primate virus species. We have used this technique to discover the macaque herpesviruses described above, as well as to identify other novel retroviruses and herpesviruses.

We are developing CODEHOP assays with broad specificity to detect both known and unknown viruses within different virus families. This approach enables the lab tests to detect low levels of virus while preserving the ability to detect known, unknown and mutated members of a virus family. CODEHOP assays have been developed to detect known and novel members of the papillomavirus, herpesvirus, adenovirus, paramyxovirus and influenzavirus families. The Rose lab is working closely with Redmond, Washington-based Micronics, Inc. to develop point-of-care microfluidics-based diagnostic tests for both paramyxovirus and influenza virus families of respiratory viruses. These tests use nucleic acid amplification and are fast, portable and affordable enough to be utilized at point of care. The paramyxovirus assay can detect and differentiate the major paramyxovirus types, including respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and parainfluenzaviruses 1-4 (PIV1-4) in a single assay.

The influenza virus assay is robust and can be used to detect both known and emerging influenza strains. This approach has the special utility of being able to detect unknown influenza strains that have pandemic capabilities, like the recent H1N1 “swine flu” and the current highly pathogenic H7N9 avian influenza strain.

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