The gene complement of wild-type human cytomegalovirus (HCMV) is incompletely understood, on account of the size and complexity of the viral genome and because laboratory strains have undergone deletions and rearrangements during adaptation to growth in culture. We have determined the sequence (241 087 bp) of chimpanzee cytomegalovirus (CCMV) and have compared it with published HCMV sequences from the laboratory strains AD169 and Toledo, with the aim of clarifying the gene content of wild-type HCMV. The HCMV and CCMV genomes are moderately diverged and essentially collinear. On the basis of conservation of potential proteincoding regions and other sequence features, we have discounted 51 previously proposed HCMV ORFs, modified the interpretations for 24 (including assignments of multiple exons) and proposed ten novel genes. Several errors were detected in the published HCMV sequences. We presently recognize 165 genes in CCMV and 145 in AD169; this compares with an estimate of 189 unique genes for AD169 made in 1990. Our best estimate for the complement of wild-type HCMV is 164 to 167 genes. INTRODUCTIONHuman cytomegalovirus (HCMV; human herpesvirus 5) is ubiquitous and largely inapparent, but poses a risk of serious disease to those lacking a competent immune system, such as neonates, transplant patients and sufferers from AIDS (reviewed in Pass, 2001). HCMV is the prototype of subfamily Betaherpesvirinae, and is the most complex of the eight human herpesvirus species. HCMV is isolated routinely on human fibroblast cell lines, and several strains in common laboratory use, such as AD169 and Towne, were derived by multiple passages on such cells (reviewed in Mocarski & Tan Courcelle, 2001).The linear, double-stranded DNA genome of AD169 comprises two covalently linked segments (L and S), each consisting of a unique region (U L and U S ) flanked by an inverted repeat (TR L and IR L , TR S and IR S ), yielding the overall genome configuration TR L -U L -IR L -IR S -U S -TR S (reviewed in Mocarski & Tan Courcelle, 2001). In addition, the genome is terminally redundant, possessing a short region (the a sequence) as a direct repeat at the termini and also in inverse orientation at the IR L -IR S junction. Some genomes contain tandemly reiterated copies of the a sequence at these locations. U L and U S can invert relative to each other by recombination between inverted repeats in replicating DNA, resulting in four equimolar genome arrangements in virion DNA. The complete DNA sequence of AD169 was published in a seminal paper by Chee et al. (1990), and at that time was the largest viral genome sequence available. The total genome size was 229 354 bp, with U L being 166 972 bp, U S 35 418 bp, R L (a collective term for TR L and IR L ) 11 247 bp, R S (TR S and IR S ) 2524 bp and the a sequence (part of R L and R S in the sizes given above) 578 bp.As a primary criterion for identifying protein-coding regions, Chee et al. (1990) focused on open reading frames (ORFs) of 100 or more contiguous amino acidencoding codons that ov...

A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus that causes the novel coronavirus disease 2019 (COVID-19), is highly transmissible and pathogenic for humans and may cause life-threatening disease and mortality, especially in individuals with underlying comorbidities. First identified in an outbreak in Wuhan, China, COVID-19 is affecting more than 185 countries and territories around the world, with more than 15,754,651 confirmed cases and more than 640,029 deaths. Since December 2019, SARS-CoV-2 transmission has become a global threat, which includes confirmed cases in all 50 states within the United States (US). As of 25 July 2020, the Johns Hopkins Whiting School of Engineering Center for Systems Science and Engineering reports more than 4,112,651 cases and 145,546 deaths. To date, health disparities are associated with COVID-19 mortality among underserved populations. Here, the author explores potential underlying reasons for reported disproportionate, increased risks of mortality among African Americans and Hispanics/Latinos with COVID-19 compared with non-Hispanic Whites. The author examines the underlying clinical implications that may predispose minority populations and the adverse clinical outcomes that may contribute to increased risk of mortality. Government and community-based strategies to safeguard minority populations at risk for increased morbidity and mortality are essential. Underserved populations living in poverty with limited access to social services across the US are more likely to have underlying medical conditions and are among the most vulnerable. Societal and cultural barriers for ethnic minorities to achieve health equity are systemic issues that may be addressed only through shifts in governmental policies, producing long-overdue, substantive changes to end health care inequities.

Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primaseassociated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.

Infection with Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is common in certain parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere, except in AIDS patients. Nevertheless, HHV8 DNA is found consistently in nearly all classical, endemic, transplant and AIDS-associated KS lesions as well as in some rare AIDS-associated lymphomas. The concept that HHV8 genomes fall into several distinct subgroups has been confirmed and refined by PCR DNA sequence analysis of the ORF-K1 gene encoding a highly variable glycoprotein related to the immunoglobulin receptor family that maps at the extreme left-hand end of the HHV-8 genome. Among more than 60 different tumor samples from the United States, central Africa, Saudi Arabia, Taiwan, and New Zealand, amino acid substitutions were found at a total of 62% of the 289 amino acid positions. These variations defined four major subtypes and 13 distinct variants or clades similar to those found for the HIV ENV protein. The B and D subtype ORF-K1 proteins differ from the A and C subtypes by 30 and 24%, respectively, whereas A and C differ from each other by 15%. In all cases tested, multiple samples from the same patient were identical. Examples of the B subtype were found almost exclusively in KS patients from Africa or of African heritage, whereas the rare D subtypes were found only in KS patients of Pacific Island heritage. In contrast, C subtypes were found predominantly in classic KS and in iatrogenic and AIDS KS in the Middle East and Asia, whereas U.S. AIDS KS samples were primarily A1, A4, and C3 variants. We conclude that this unusually high diversity, in which 85% of the nucleotide changes lead to amino acid changes, reflects some unknown powerful biological selection process that has been acting preferentially on this early lytic cycle membrane signalling protein. Two distinct levels of ORF-K1 variability are recognizable. Subtype-specific variability indicative of long-term evolutionary divergence is both spread throughout the protein as well as concentrated within two 40-amino-acid extracellular domain variable regions (VR1 and VR2), whereas intratypic variability localizes predominantly within a single 25-amino-acid hypervariable Cys bridge loop and apparently represents much more recent changes that have occurred even within specific clades. In contrast, numerous extracellular domain glycosylation sites and Cys bridge residues as well as the ITAM motif in the cytoplasmic domain are fully conserved. Overall, we suggest that rather than being a newly acquired human pathogen, HHV8 is an ancient human virus that is preferentially transmitted in a familial fashion and is difficult to transmit horizontally in the absence of immunosuppression. The division into the four major HHV8 subgroups is probably the result of isolation and founder effects associated with the history of migration of modern human populations out of Africa over the past 35,000 to 60,000 years.

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)-alpha-like receptor gene, the US28 beta-chemokine 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 (P=.04). There was no association between specific subtypes of the US28 and UL55 genes and outcome (P=.864 and P=.765, respectively). Multiple genotypes (implying multiple infections) were detected in tissues from 8 of 10 autopsies. Therefore, polymorphism in the CMV-encoded TNF-alpha-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.

BackgroundCongenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood–brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.MethodsPrimary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated ‘SBCMV’. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.ResultsHCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.ConclusionHCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.

Kaposi’s sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0.35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized “orphan” region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.