Jian Chao Zong

Patterns of Gene Expression and a Transactivation Function Exhibited by the vGCR (ORF74) Chemokine Receptor Protein of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT The ORF74 or vGCR gene encoded by Kaposis sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) has properties of a ligand-independent membrane receptor signaling protein with angiogenic properties that is predicted to play a key role in the biology of the virus. We have examined the expression of vGCR mRNA and protein in primary effusion lymphoma (PEL) cell lines, PEL and multicentric Castlemans disease (MCD) tumors, Kaposis sarcoma lesions and infected endothelial cell cultures. The vGCR gene proved to be expressed in PEL cell lines as a large spliced bicistronic mRNA of 3.2 kb that also encompasses the upstream vOX2 (K14) gene. This mRNA species was induced strongly by phorbol ester (TPA) and sodium butyrate treatment in the BCBL-1 cell line, but only weakly in the HBL6 cell line, and was classified as a relatively late and low-abundance delayed early class lytic cycle gene product. A complex bipartite upstream lytic cycle promoter for this mRNA was nestled within the intron of the 5′-overlapping but oppositely oriented latent-state transcription unit for LANA1/vCYC-D/vFLIP and responded strongly to both TPA induction and cotransfection with the KSHV RNA transactivator protein (RTA or ORF50) in transient reporter gene assays. A vGCR protein product of 45 kDa that readily dimerized was detected by Western blotting and in vitro translation and was localized in a cytoplasmic and membrane pattern in DNA-transfected Vero and 293T cells or adenovirus vGCR-transduced dermal microvascular endothelial cells (DMVEC) as detected by indirect immunofluorescence assay (IFA) and immunohistochemistry with a specific rabbit anti-vGCR antibody. Similarly, a subfraction of KSHV-positive cultured PEL cells and of KSHV (JSC-1) persistently infected DMVEC cells displayed cytoplasmic vGCR protein expression, but only after TPA or spontaneous lytic cycle induction, respectively. The vGCR protein was also detectable by immunohistochemical staining in a small fraction (0.5 to 3%) of the cells in PEL and MCD tumor and nodular Kaposis sarcoma lesion specimens that were apparently undergoing lytic cycle expression. These properties are difficult to reconcile with the vGCR proteins playing a direct role in spindle cell proliferation, transformation, or latency, but could be compatible with proposed contributions to angiogenesis via downstream paracrine effects. The ability of vGCR to transactivate expression of both several KSHV promoter-driven luciferase (LUC) reporter genes and an NFκB motif containing the chloramphenicol acetyltransferase (CAT) reporter gene may also suggest an unexpected regulatory role in viral gene expression.

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

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)

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 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 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

OBJECTIVE 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. ANIMALS 5 apparently healthy captive Asian elephants (Elephas maximus) from the same herd. PROCEDURES 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. RESULTS 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. CONCLUSIONS AND CLINICAL RELEVANCE 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.

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.

Ugandan Kaposi’s Sarcoma-Associated Herpesvirus Phylogeny: Evidence for Cross-Ethnic Transmission of Viral Subtypes

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 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.

KINETICS OF VIRAL LOADS AND GENOTYPIC ANALYSIS OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS-1 INFECTION IN CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract:  Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elephas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3–8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7–21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

FATAL HERPESVIRUS HEMORRHAGIC DISEASE IN WILD AND ORPHAN ASIAN ELEPHANTS IN SOUTHERN INDIA

Up to 65% of deaths of young Asian elephants (Elephas maximus) between 3 mo and 15 yr of age in Europe and North America over the past 20 yr have been attributed to hemorrhagic disease associated with a novel DNA virus called elephant endotheliotropic herpesvirus (EEHV). To evaluate the potential role of EEHV in suspected cases of a similar lethal acute hemorrhagic disease occurring in southern India, we studied pathologic tissue samples collected from field necropsies. Nine cases among both orphaned camp and wild Asian elephants were identified by diagnostic PCR. These were subjected to detailed gene subtype DNA sequencing at multiple PCR loci, which revealed seven distinct strains of EEHV1A and one of EEHV1B. Two orphan calves that died within 3 days of one another at the same training camp had identical EEHV1A DNA sequences, indicating a common epidemiologic source. However, the high level of EEHV1 subtype genetic diversity found among the other Indian strains matches that among over 30 EEHV1 strains that have been evaluated from Europe and North America. These results argue against the previous suggestions that this is just a disease of captive elephants and that the EEHV1 virus has crossed recently from African elephant (Loxodonta africana) hosts to Asian elephants. Instead, both the virus and the disease are evidently widespread in Asia and, despite the disease severity, Asian elephants appear to be the ancient endogenous hosts of both EEHV1A and EEHV1B.

Elephant endotheliotropic herpesviruses EEHV1A, EEHV1B, and EEHV2 from cases of hemorrhagic disease are highly diverged from other mammalian herpesviruses and may form a new subfamily.

ABSTRACT A family of novel endotheliotropic herpesviruses (EEHVs) assigned to the genus Proboscivirus have been identified as the cause of fatal hemorrhagic disease in 70 young Asian elephants worldwide. Although EEHV cannot be grown in cell culture, we have determined a total of 378 kb of viral genomic DNA sequence directly from clinical tissue samples from six lethal cases and two survivors. Overall, the data obtained encompass 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus 10 novel genes, including a single large putative transcriptional regulatory protein (ORF-L). On the basis of differences in gene content and organization plus phylogenetic analyses of conserved core proteins that have just 20% to 50% or less identity to orthologues in other herpesviruses, we propose that EEHV1A, EEHV1B, and EEHV2 could be considered a new Deltaherpesvirinae subfamily of mammalian herpesviruses that evolved as an intermediate branch between the Betaherpesvirinae and Gammaherpesvirinae. Unlike cytomegaloviruses, EEHV genomes encode ribonucleotide kinase B subunit (RRB), thymidine kinase (TK), and UL9-like origin binding protein (OBP) proteins and have an alphaherpesvirus-like dyad symmetry Ori-Lyt domain. They also differ from all known betaherpesviruses by having a 40-kb large-scale inversion of core gene blocks I, II, and III. EEHV1 and EEHV2 DNA differ uniformly by more than 25%, but EEHV1 clusters into two major subgroups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three nonadjacent loci totaling 15 kb diverge by between 21 and 37%. One strain of EEHV1B analyzed is interpreted to be a modern partial recombinant with EEHV1A. IMPORTANCE Asian elephants are an endangered species whose survival is under extreme pressure in wild range countries and whose captive breeding populations in zoos are not self-sustaining. In 1999, a novel class of herpesviruses called EEHVs was discovered. These viruses have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the United States and Europe since 1980. The disease is increasingly being recognized in Asian range countries as well. These viruses cannot be grown in cell culture, but by direct PCR DNA sequence analysis from segments totaling 15 to 30% of the genomes from blood or necropsy tissue from eight different cases, we have determined that they fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, and we propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

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.