Gary S Hayward

Publications of Gary S Hayward

• Comparison of humoral immune responses to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus using a viral proteome microarray.

  Authors: Dasheng Zheng, Jun Wan, Yong Gu Cho, Leyao Wang, Chuang-Jiun Chiou, Shweta Pai, Crystal Woodard, Jian Zhu, Gangling Liao, Otoniel Martinez-Maza, Jiang Qian, Heng Zhu, Gary S Hayward, Richard F Ambinder, S Diane Hayward

  The Journal of infectious diseases. 12/2011; 204(11):1683-91.

  Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have anEpstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi's sarcoma or lymphoma. In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1-positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

• Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication.

  Authors: Renfeng Li, Jian Zhu, Zhi Xie, Gangling Liao, Jianyong Liu, Mei-Ru Chen, Shaohui Hu, Crystal Woodard, Jimmy Lin, Sean D Taverna, Prashant Desai, Richard F Ambinder, Gary S Hayward, Jiang Qian, Heng Zhu, S Diane Hayward

  Cell host & microbe. 10/2011; 10(4):390-400.

  Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they eachHerpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies.

• KSHV regulation of fibulin-2 in Kaposi's sarcoma: implications for tumorigenesis.

  Authors: Donald J Alcendor, Susan Knobel, Prashant Desai, Wen Qui Zhu, Gary S Hayward

  The American journal of pathology. 09/2011; 179(3):1443-54.

  Kaposi's sarcoma is an angioproliferative tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection of vascular endothelial cells. Fibulins, proteins that associate with extracellularKaposi's sarcoma is an angioproliferative tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection of vascular endothelial cells. Fibulins, proteins that associate with extracellular matrix (ECM) proteins, may have both tumor-suppressive and oncogenic activities. We found that the expression of fibulin-2 protein and mRNA were decreased 50-fold and 26-fold, respectively, in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC). Using quantitative RT-PCR, we found a fivefold and 25-fold decrease of fibulin-2 extracellular matrix binding partners, fibronectin and tropoelastin, respectively. Time-course transcriptional analyses over 10 days showed that in addition to that of fibulin-2, expression of fibulins 3 and 5 was decreased in KSHV-infected DMVEC, fibulins 1C/1D were increased, and fibulins 4, 6, and 7 were unchanged. KSHV latency-associated nuclear antigen (LANA) transcription levels rose consistently over the same period. Addition of recombinant fibulin-3 or -5 for 48 hours to 10-day KSHV-infected cells caused a suppression of KSHV-induced vascular endothelial growth factor (VEGF) protein and mRNA levels. Recombinant fibulin-3 also significantly reduced VEGF receptor 3 expression. In pleural effusion lymphoma cell lines that express variable levels of KSHV lytic replication, we observed no detectable fibulin-2 or -5 expression. Finally, fibulin-2 expression was decreased in tissue microarrays from KSHV-infected, LANA-positive patient cells as compared to that in patient nontumor controls. Understanding the interactions between KSHV and the fibulins may lead to the development of novel therapies for treatment of Kaposi's sarcoma.

• Detection and evaluation of novel herpesviruses in routine and pathological samples from Asian and African elephants: identification of two new probosciviruses (EEHV5 and EEHV6) and two new gammaherpesviruses (EGHV3B and EGHV5).

  Authors: Erin Latimer, Jian-Chao Zong, Sarah Y Heaggans, Laura K Richman, Gary S Hayward

  Veterinary microbiology. 01/2011; 147(1-2):28-41.

  Systemic infections with elephant endotheliotropic herpesviruses (EEHV) cause a rapid onset acute hemorrhagic disease with an 85% mortality rate. More than 60 cases have been confirmed worldwideSystemic infections with elephant endotheliotropic herpesviruses (EEHV) cause a rapid onset acute hemorrhagic disease with an 85% mortality rate. More than 60 cases have been confirmed worldwide occurring predominantly in juvenile Asian elephants. Originally, three virus types EEHV1A, EEHV1B and EEHV2 were identified, all members of the Proboscivirus genus within the Betaherpesvirinae. However, four elephant gammaherpesviruses (EGHV) have also been found by DNA PCR approaches in eye and genital secretions of asymptomatic animals, and two more versions of the probosciviruses, EEHV3 and EEHV4, were recently detected in acute hemorrhagic disease cases. To ask whether even more species of elephant herpesviruses may exist, we have developed several new diagnostic DNA PCR assays using multiple round primers in the DNA POL region. These have been used routinely for nearly three years to screen samples submitted to the Elephant Herpesvirus Laboratory for diagnosis of possible cases of EEHV disease in blood and necropsy tissue, as well as in biopsies of other suspicious lesions or growths. Several more cases of EEHV1-associated hemorrhagic disease were confirmed, but in addition, we describe here eleven examples of other known and novel herpesviruses detected and evaluated with these reagents. They include the prototypes of four new elephant herpesviruses, two more within the proboscivirus group EEHV5 and EEHV6, plus two more gammaherpesviruses EGHV3B and EGHV5. We also report initial semi-quantitative PCR assays demonstrating very high viral loads in the blood of the EEHV3 and EEHV4-associated hemorrhagic disease cases.

• Detection of a single identical cytomegalovirus (CMV) strain in recently seroconverted young women.

  Authors: Suchetha Murthy, Gary S Hayward, Sarah Wheelan, Michael S Forman, Jin-Hyun Ahn, Robert F Pass, Ravit Arav-Boger

  PloS one. 01/2011; 6(1):e15949.

  Infection with multiple CMV strains is common in immunocompromised hosts, but its occurrence in normal hosts has not been well-studied. We analyzed CMV strains longitudinally in women who acquiredInfection with multiple CMV strains is common in immunocompromised hosts, but its occurrence in normal hosts has not been well-studied. We analyzed CMV strains longitudinally in women who acquired CMV while enrolled in a CMV glycoprotein B (gB) vaccine trial. Sequencing of four variable genes was performed in samples collected from seroconversion and up to 34 months thereafter. 199 cultured isolates from 53 women and 65 original fluids from a subset of 19 women were sequenced. 51 women were infected with one strain each without evidence for genetic drift; only two women shed multiple strains. Genetic variability among strains increased with the number of sequenced genetic loci. Nevertheless, 13 of 53 women proved to be infected with an identical CMV strain based on sequencing at all four variable genes. CMV vaccine did not alter the degree of genetic diversity amongst strains. Primary CMV infection in healthy women nearly always involves shedding of one strain that remains stable over time. Immunization with CMVgB-1 vaccine strain is not selective against specific strains. Although 75% of women harbored their unique strain, or a strain shared with only one other woman, 25% shared a single common strain, suggesting that this predominant strain with a particular combination of genetic loci is advantageous in this large urban area.

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

  Authors: Ran He, Gordon Sandford, Gary S Hayward, William H Burns, Gary H Posner, Michael Forman, Ravit Arav-Boger

  Virology journal. 01/2011; 8(1):40.

  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 ofRecombinant 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.

• The ubiquitin E3 ligase RAUL negatively regulates type i interferon through ubiquitination of the transcription factors IRF7 and IRF3.

  Authors: Yanxing Yu, Gary S Hayward

  Immunity. 12/2010; 33(6):863-77.

  In the course of combating infectious agents, type I interferon (IFN) needs a timely downregulation mechanism to avoid detrimental overreaction. Here we showed a mechanism for restraining type I IFNIn the course of combating infectious agents, type I interferon (IFN) needs a timely downregulation mechanism to avoid detrimental overreaction. Here we showed a mechanism for restraining type I IFN responses, which relied on a HECT domain ubiquitin (Ub) E3 ligase, RAUL. RAUL limited type I IFN production by directly catalyzing lysine 48-linked polyubiquitination of both interferon regulatory factor 7 (IRF7) and IRF3 followed by proteasome-dependent degradation. Suppression of RAUL by dominant-negative RAUL or siRNA augmented both basal and virus-induced production of type I IFN, which resulted in reduced viral replication. The Kaposi's sarcoma-associated herpes virus immediate-early lytic cycle trigger protein RTA recruited this mechanism to augment its countermeasures against the host antiviral response. These results unveil a previously unrecognized "brake mechanism" for type I IFN that maintains proper low amounts of type I IFN under physiological conditions and restrains its magnitude when the antiviral response intensifies.

• Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay.

  Authors: Jeffrey J Stanton, Jian-Chao Zong, Erin Latimer, Jie Tan, Alan Herron, Gary S Hayward, Paul D Ling

  American journal of veterinary research. 08/2010; 71(8):925-33.

  To investigate the pathogenesis and transmission of elephant endotheliotropic herpesvirus (EEHV1) by analyzing various elephant fluid samples with a novel EEHV1-specific real-time PCR assay. 5To investigate the pathogenesis and transmission of elephant endotheliotropic herpesvirus (EEHV1) by analyzing various elephant fluid samples with a novel EEHV1-specific real-time PCR assay. 5 apparently healthy captive Asian elephants (Elephas maximus) from the same herd. A real-time PCR assay was developed that specifically detects EEHV1. The assay was used to evaluate paired whole blood and trunk-wash samples obtained from the 5 elephants during a 15-week period. Deoxyribonucleic acid sequencing and viral gene subtyping analysis were performed on trunk-wash DNA preparations that had positive results for EEHV1. Viral gene subtypes were compared with those associated with past fatal cases of herpesvirus-associated disease within the herd. The PCR assay detected viral DNA to a level of 1,200 copies/mL of whole blood. It was used to detect EEHV1 in trunk secretions of 3 of the 5 elephants surveyed during the 15-week period. Viral gene subtyping analysis identified 2 distinct elephant herpesviruses, 1 of which was identical to the virus associated with a previous fatal case of herpesvirus-associated disease within the herd. EEHV1 was shed in the trunk secretions of healthy Asian elephants. Trunk secretions may provide a mode of transmission for this virus. Results of this study may be useful for the diagnosis, treatment, and management of EEHV1-associated disease and the overall management of captive elephant populations.

• KSHV downregulation of galectin-3 in Kaposi's sarcoma.

  Authors: Donald J Alcendor, Susan M Knobel, Prashant Desai, Wen Q Zhu, Hollie E Vigil, Gary S Hayward

  Glycobiology. 05/2010; 20(5):521-32.

  Galectins are a family of proteins that share an affinity for beta-galactoside containing glycoconjugates. In prostate, ovarian and breast cancer, downregulation of galectin-3 is associated withGalectins are a family of proteins that share an affinity for beta-galactoside containing glycoconjugates. In prostate, ovarian and breast cancer, downregulation of galectin-3 is associated with malignancy and tumor progression. Kaposi's sarcoma (KS) is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of primary effusion lymphomas and some forms of multicentric Castleman's disease. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS. We found reduced levels of galectin-3 expression in a significant fraction of latency-associated nuclear antigen (LANA)-positive spindle cell regions in human archival KS tissue and as measured in KS tissue microarrays. Here we demonstrate that galectin-3 protein expression is downregulated 10-fold in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC) accompanied by downregulation of message. There is loss of galectin-3 staining in KSHV-infected DMVEC by dual labeled immunohistochemistry in LANA-positive spindle cells. We observed a consistent downregulation of galectin-3 by time-course transcriptional analysis. Of the galectins assayed, only galectin-1 was also downregulated in KSHV-infected DMVEC. We examined 86 KS tumors; 19 were LANA positive (22%) and 67 LANA negative (78%). All 86 tumors were found to be galectin-3 positive; 11 of 19 showed reduced expression of galectin-3 in LANA-positive spindle cell regions. Our data suggest that KSHV vFLIP and LANA are the viral genes targeting galectin-3 downregulation. The contribution of host factors to the pathogenesis of KS is essential for early detection and development of innovative therapies for treatment.

• Deletion of the rat cytomegalovirus immediate early 1 gene results in a virus capable of establishing latency but with lower levels of acute virus replication and latency that compromise reactivation efficiency.

  Authors: Gordon R. Sandford, Uwe Schumacher, Jakob Ettinger, Wolfram Brune, Gary S Hayward, William H Burns, Sebastian Voigt

  The Journal of general virology. 11/2009;

  The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection.The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection. Whilst IE2 is essential for triggering the lytic cycle, the exact roles of IE1 have not been resolved. An MIE-exon 4 deleted rat cytomegalovirus (DeltaIE1) failed to synthesize the IE1 protein and did not disperse promyelocytic leukemia bodies (PML) early post-infection, but it was still capable of normal replication in fibroblast cell culture. However, DeltaIE1 had diminished ability to infect salivary glands persistently in vivo and to reactivate from spleen explant cultures ex vivo. Quantitation of viral genomes in spleens of infected animals revealed a reduced amount of DeltaIE1 virus produced during acute infection, suggesting a role for IE1 as a regulator in establishing a chronic or persistent infection, rather than in more directly influencing the latency or reactivation processes.

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• Patterns of divergence in the vCXCL and vGPCR gene clusters in primate cytomegalovirus genomes.

  Authors: Donald J Alcendor, Jianchao Zong, Aidan Dolan, Derek Gatherer, Andrew J Davison, Gary S Hayward

  Virology. 10/2009;

  Primate cytomegalovirus (CMV) genomes contain tandemly repeated gene clusters putatively encoding divergent CXC chemokine ligand-like proteins (vCXCLs) and G protein-coupled receptor-like proteinsPrimate cytomegalovirus (CMV) genomes contain tandemly repeated gene clusters putatively encoding divergent CXC chemokine ligand-like proteins (vCXCLs) and G protein-coupled receptor-like proteins (vGPCRs). In human, chimpanzee and rhesus CMVs, respectively, the vCXCL cluster contains two, three and six genes, and the vGPCR cluster contains two, two and five genes. We report that (i) green monkey CMV strains fall into two groups, containing either eight and five genes or seven and six genes in the respective clusters, and (ii) owl monkey CMV has two and zero genes. Phylogenetic analysis suggested that the vCXCL cluster evolved from a CXCL chemokine gene (probably GRO-alpha) that was captured in an incompletely spliced form by an ancestor of Old and New World primate CMVs, and that the vGPCR cluster evolved from a GPCR gene captured by an Old World primate CMV. Both clusters appear to have evolved via complex duplication and deletion events.

• Effects of Chemotherapy in AIDS-Associated Non-Hodgkin's Lymphoma on Kaposi's Sarcoma Herpesvirus DNA in Blood.

  Authors: Lan Lin, Jeannette Y Lee, Lawrence D Kaplan, Bruce J Dezube, Ariela Noy, Susan E Krown, Alexandra M Levine, Yanxing Yu, Gary S Hayward, Richard F Ambinder

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 05/2009;

  PURPOSE: To determine the relative frequency with which Kaposi's sarcoma-associated herpesvirus/HHV-8 (KSHV) DNA is detected in peripheral-blood mononuclear cells (PBMCs) and in plasma of patientsPURPOSE: To determine the relative frequency with which Kaposi's sarcoma-associated herpesvirus/HHV-8 (KSHV) DNA is detected in peripheral-blood mononuclear cells (PBMCs) and in plasma of patients with AIDS-KS and AIDS-associated non-Hodgkin's lymphoma (NHL; AIDS-NHL); to determine whether the presence of viral DNA in plasma reflects lysis of tumor cells or reflects the presence of viremia (ie, virion-encapsidated DNA); and to determine the effect of lymphoma therapy on KSHV DNA. PATIENTS AND METHODS: Samples were obtained from patients enrolled in AIDS Malignancy Consortium clinical trials and from healthy donors. Real time PCR was used to quantify KSHV DNA in peripheral blood mononuclear cells (PBMC) and plasma. DNase digestion and fragment size determination studies were used to characterize the DNA detected. RESULTS: In patients with AIDS-KS, KSHV DNA was detected in PBMC (54%) and in plasma (62%). In patients with AIDS-NHL, KSHV DNA was detected in PBMC (19%) and in plasma (22%). Median copy numbers also differed. KSHV DNA in plasma appeared to be encapsidated. In six patients with AIDS-NHL who were treated with chemotherapy (with or without rituximab), KSHV copy number declined in PBMC and in plasma. CONCLUSION: KSHV DNA is sometimes detected in PBMC or in plasma of patients with AIDS-NHL without KS. Among patients with KSHV DNA detected in PBMC or in plasma, copy number does not distinguish between patients with AIDS-NHL and AIDS-KS. KSHV DNA in plasma likely reflects viremia and not simply lysis of tumor or other KSHV-infected cells. KSHV DNA copy number in PBMC and in plasma declined with lymphoma-directed cytotoxic chemotherapy in each of the six patients studied.

• Protein Array Identification of Substrates of the Epstein-Barr Virus Protein Kinase BGLF4.

  Authors: Jian Zhu, Gangling Liao, Liang Shan, Jun Zhang, Mei-Ru Chen, Gary S Hayward, S Diane Hayward, Prashant Desai, Heng Zhu

  Journal of virology. 03/2009;

  A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeuticA conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an EBV protein array to evaluate the targets of the EBV protein kinase, BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrecognized substrates for BGLF4, illustrating the broad role played by this protein kinase. Approximately half of the BGLF4 targets were also in vitro substrates for the cellular kinase CDK1/Cyclin B. Unexpectedly, EBNA1 was identified as a substrate and binding partner of BGLF4. EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency. BGLF4 did not prevent EBNA1 binding to sites in the EBV latency origin of replication oriP. Rather, we found that BGLF4 was recruited by EBNA1 to oriP in cells transfected with an oriP vector and BGLF4 and in lytically induced EBV positive Akata cells. In cells transfected with an oriP vector, the presence of BGLF4 led to more rapid loss of the episomal DNA and this was dependent on BGLF4 kinase activity. Similarly, expression of dox-inducible BGLF4 in Akata cells led to a reduction in episomal EBV genomes. We propose that BGLF4 contributes to effective EBV lytic cycle progression not only through phosphorylation of EBV lytic DNA replication and virion proteins but also by interfering with EBNA1 replication function.

• Binding STAT2 by the acidic domain of human cytomegalovirus IE1 promotes viral growth and is negatively regulated by SUMO.

  Authors: Yong Ho Huh, Young-Eui Kim, Eui Tae Kim, Jung-Jin Park, Moon Jung Song, Hua Zhu, Gary S Hayward, Jin-Hyun Ahn

  Journal of virology. 09/2008;

  The human cytomegalovirus (HCMV) 72-kDa immediate-early (IE) 1 protein is thought to modulate cellular antiviral functions impacting on PML nuclear bodies and signal transducer and activator ofThe human cytomegalovirus (HCMV) 72-kDa immediate-early (IE) 1 protein is thought to modulate cellular antiviral functions impacting on PML nuclear bodies and signal transducer and activator of transcription (STAT) signaling. IE1 consists of four distinct regions - an amino-terminal region required for nuclear localization, a large central hydrophobic region responsible for PML targeting and transactivation activity, an acidic domain, and a carboxyl-terminal chromatin tethering domain. We found that the acidic domain of IE1 is required for binding to STAT2. A mutant HCMV encoding the acidic domain-deleted IE1(Delta421-475) was generated. In mutant virus-infected cells, IE1(Delta421-475) failed to bind to STAT2. The growth of mutant virus was only slightly delayed at high multiplicity of infection (MOI), but was severely impaired at low MOI with low level accumulation of viral proteins. When cells were pretreated with interferon beta, the mutant virus showed an additional 1,000-fold reduction in viral growth even at high MOI, compared to wild-type. The inhibition of STAT2 loading on the target promoter upon infection was markedly reduced with mutant virus. Furthermore, sumoylation of IE1 at this acidic domain was found to abolish the activity of IE1 to bind to STAT2 and repress the interferon-stimulated genes. Our results provide genetic evidence that IE1 binding to STAT2 requires the 55-amino acid acidic domain and promotes viral growth by interfering with interferon signaling, and demonstrate that this viral activity is negatively regulated by a cellular sumoylation pathway.

• Reflections on the interpretation of heterogeneity and strain differences based on very limited PCR sequence data from Kaposi's sarcoma-associated herpesvirus genomes.

  Authors: Jian-Chao Zong, Ravit Arav-Boger, Donald J Alcendor, Gary S Hayward

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 10/2007; 40(1):1-8.

  Ever since the original identification of fragments of KSHV DNA in Kaposi's sarcoma (KS) tissue by Chang et al. in 1994, PCR has been used successfully and extensively to detect the virus in clinicalEver since the original identification of fragments of KSHV DNA in Kaposi's sarcoma (KS) tissue by Chang et al. in 1994, PCR has been used successfully and extensively to detect the virus in clinical samples from the accepted etiological diseases of KS, PEL and MCD. However, a number of other clinical and epidemiological studies claiming evidence for KSHV in multiple myeloma or sarcoid and more recently in primary pulmonary hypertension, as well as claims about the biological significance of DNA sequence polymorphisms based just on small ORF26 PCR DNA fragments have not been convincing. Here, we evaluate the validity and interpretations of previous results in the context of both the observed rates and global patterns of sequence variability within an extended ORF26 locus, as well as from the perspective of the overall levels of KSHV variability found after sampling multiple loci across the complete KSHV genome. The results cast doubts on most claims for biological significance for these polymorphisms, which instead correlate with viral subtype clustering arising from geographic and ethnic divergence of the ancestral human hosts. In addition, we describe several observations that help to explain likely sources of the often either unexpectedly high or unexpectedly low levels of sporadic variability seen in the PCR DNA sequence data reported in some of those studies.

• Evaluation of global clustering patterns and strain variation over an extended ORF26 gene locus from Kaposi's sarcoma herpesvirus.

  Authors: Jian-Chao Zong, Henry Kajumbula, William Boto, Gary S Hayward

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 10/2007; 40(1):19-25.

  BACKGROUND: Small 233-bp or 330-bp DNA fragments of the ORF26 gene of human Kaposi's sarcoma herpesvirus (KSHV) have been used extensively to identify KSHV by PCR in clinical samples; to associateBACKGROUND: Small 233-bp or 330-bp DNA fragments of the ORF26 gene of human Kaposi's sarcoma herpesvirus (KSHV) have been used extensively to identify KSHV by PCR in clinical samples; to associate KSHV with novel diseases and to correlate KSHV strain differences with pathogenicity. OBJECTIVES: We evaluated the nature, extent and source of nucleotide sequence variability among a large and diverse set of known KSHV-positive DNA samples. STUDY DESIGN: Direct DNA PCR sequencing was carried out on 136 distinct Kaposi's sarcoma and primary effusion lymphoma-related samples from different geographic locations. RESULTS: The presence of 26 diagnostic nucleotide polymorphisms across an expanded 965-bp PCR locus define eight distinct ORF26E genotypes, three being of Eurasian origin, one from the Pacific Rim, and five from Sub-Saharan Africa. Previous ambiguities between some genotype patterns in the 330-bp locus data are fully resolved. CONCLUSIONS: This analysis provides an expanded database for understanding and evaluating ORF26 polymorphisms. In particular, the eight genotype clusters correlated with specific ethnic and geographic origins of the patients. Furthermore, the very low level of additional sporadic nucleotide variation found permits detection of spurious sequence errors or contamination present in some published data.

• Cytomegalovirus (CMV)-encoded UL144 (truncated tumor necrosis factor receptor) and outcome of congenital CMV infection.

  Authors: Ravit Arav-Boger, Catherine A Battaglia, Tiziana Lazzarotto, Liliana Gabrielli, Jian C Zong, Gary S Hayward, Marie Diener-West, Maria P Landini

  The Journal of infectious diseases. 09/2006; 194(4):464-73.

  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 polymorphismsBACKGROUND: 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.

• Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells.

  Authors: Jian Huang, Gangling Liao, Honglin Chen, Frederick Y Wu, Lindsey Hutt-Fletcher, Gary S Hayward, S Diane Hayward

  Journal of virology. 03/2006; 80(3):1098-109.

  The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressedThe contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.

• Ugandan Kaposi's sarcoma-associated herpesvirus phylogeny: evidence for cross-ethnic transmission of viral subtypes.

  Authors: Henry Kajumbula, Robert G Wallace, Jian-Chao Zong, Joseph Hokello, Noah Sussman, Simon Simms, Robert F Rockwell, Robert Pozos, Gary S Hayward, William Boto

  Intervirology. 02/2006; 49(3):133-43.

  OBJECTIVE: The aim of this study was to test the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) phylogeny and host ethnicity at the within-country scale. METHODS: KSHV genomicOBJECTIVE: The aim of this study was to test the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) phylogeny and host ethnicity at the within-country scale. METHODS: KSHV genomic DNA samples were isolated from 31 patients across eleven Ugandan ethnic groups. Amino acid sequences of the ORF-K1 gene were used to construct a neighbor-joining phylogenetic tree. RESULTS: A5 and B1 variants predominated with no evidence of distinct ethnic or geographic distribution. A new K1 subtype (F) was identified in a member of the Bantu Gisu tribe and a new subtype B variant (B3) among members of the Bantu Ganda tribe. CONCLUSIONS: The phylogeny may yet be structured by host ethnicity if members of Ugandan groups have convoluted biological origins, even as they identify with single tribes. An alternative possibility is that KSHV subtype evolution may have preceded major diversification of sub-Saharan Africans into ethnicities as we know them today, with ethnic groups beginning their histories already hosting multiple subtypes. A third alternative is that horizontal transmission of multiple KSHV subtypes may have broken up vertical lineages of the virus passed down within Ugandan populations.

• The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht.

  Authors: Sebastian Voigt, Gordon R. Sandford, Gary S Hayward, William H Burns

  The Journal of general virology. 03/2005; 86(Pt 2):263-74.

  Sequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the majorSequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the major immediate-early (MIE) genes of the English isolate of rat cytomegalovirus (RCMV), are presented. The results identified important differences compared to both murine cytomegalovirus (MCMV) and the Maastricht isolate of RCMV. A striking finding is the presence of a highly conserved, rightwards-oriented homologue of the rat cellular CD200 (OX2) gene immediately to the right of the MIE region, which replaces either the leftwards-oriented AAV REP gene of RCMV (Maastricht) or the upstream spliced portions of the immediate-early 2 gene (ie2) in MCMV. From the presence of other homologues of MCMV- and RCMV-specific genes, such as the beta-chemokine MCK-2, SGG1 and an Fcgamma receptor gene, as reported here, the basic architecture of the MIE region (reported previously) and the level of IE2 and DNA polymerase (POL) protein conservation in phylogenetic analyses, it is clear that the English strain of RCMV is also a member of the genus Muromegalovirus, but is a beta-herpesvirus species that is very distinct from both MCMV and RCMV (Maastricht). Both the lack of a CD200 homologue in the other two rodent viruses and the depth of sequence divergence of the rodent CMV IE2 and POL proteins suggest that these three viruses have evolved as separate species in the genus Muromegalovirus since very early in the host rodent lineage.