Herpesviridae

Herpesviruses are successful pathogens that infect most vertebrates as well as at least one invertebrate species. Six of the eight human herpesviruses are widely distributed in the population. Herpesviral infections persist for the life of the infected host due in large part to the ability of these viruses to enter a non-productive, latent state in which viral gene expression is limited and immune detection and clearance is avoided. Periodically, the virus will reactivate and enter the lytic cycle, producing progeny virus that can spread within or to new hosts. Latency has been classically divided into establishment, maintenance, and reactivation phases. Here we focus on demonstrated and postulated molecular mechanisms leading to the establishment of latency for representative members of each human herpesvirus family. Maintenance and reactivation are also briefly discussed. In particular, the roles that tegument proteins may play during latency are highlighted. Finally, we introduce the term animation to describe the initiation of lytic phase gene expression from a latent herpesvirus genome, and discuss why this step should be separated, both molecularly and theoretically, from reactivation.

Epstein-Barr virus (EBV) is a highly successful herpesvirus, colonizing more than 90% of the adult human population worldwide, although it is also associated with various malignant diseases. Primary infection is usually clinically silent, and subsequent establishment of latency in the memory B lymphocyte compartment allows persistence of the virus in the infected host for life. EBV is so markedly B-lymphotropic when exposed to human lymphocytes in vitro that the association of EBV with rare but distinct types of T and NK cell lymphoproliferations was quite unexpected. Whilst relatively rare, these EBV-associated T and NK lymphoproliferations can be therapeutically challenging and prognosis for the majority of patients is dismal. In this review, we summarize the current knowledge on the role of EBV in the pathogenesis of these tumours, and the implications for treatment.

Innate recognition is essential in the antiviral response against infection by herpes simplex virus (HSV). Chemokines are important for control of HSV via recruitment of natural killer cells, T lymphocytes, and antigen-presenting cells. We previously found that early HSV-1-mediated chemokine responses are not dependent on TLR2 and TLR9 in human macrophages. Here, we investigated the role of the recently identified innate IFN-inducible DNA receptor IFI16 during HSV-1 infection in human macrophages. Peripheral blood mononuclear cells were purified from buffy coats and monocytes were differentiated to macrophages. Macrophages infected with HSV-1 were analyzed using siRNA-mediated knock-down of IFI16 by real-time PCR, ELISA, and Western blotting. We determined that both CXCL10 and CCL3 are induced independent of HSV-1 replication. IFI16 mediates CCL3 mRNA accumulation during early HSV-1 infection. In contrast, CXCL10 was induced independently of IFI16. Our data provide the first evidence of HSV-1-induced innate immune responses via IFI16 in human primary macrophages. In addition, the data suggest that at least one additional unidentified receptor or innate sensing mechanism is involved in recognizing HSV-1 prior to viral replication.

Diagnosis of cytomegalovirus (CMV) infection is challenging because of the high rate of asymptomatic infection and the low specificity of associated symptoms and signs. As a result, laboratory testing is an essential aid in making an accurate diagnosis. The presence of CMV IgM is indicative of primary CMV infection. In pregnancy, diagnosis of primary infection is important because primary maternal infection increases fetal infection risk substantially. Fetal infection can result in serious sequelae ranging from neurological deficits to death. Diagnosis among the immunocompromised is also critical for the timely initiation of therapy that can reduce morbidity and mortality risk. The IMMULITE® 2000 CMV IgM assay qualitatively detects CMV IgM antibodies in human serum or plasma to aid in the diagnosis of current or recent CMV infection. To determine expected values in apparently healthy subjects, 136 samples were tested. Reproducibility, normal range, and method comparison studies were also performed to evaluate the assay's performance. The assay's reproducibility was evaluated across three sites. Seven hundred and eighteen (n = 718) individual patient serum samples, which included samples from CMV IgM-positive (n = 109, determined by the Abbott IMx CMV or the Diamedix CMV IgM assays), pregnant (n = 210), HIV-positive (n = 30), immunosuppressed (n = 102), and transplant patients (n = 17) and from patients with potentially cross-reacting conditions (n = 136) were evaluated in the method comparison study. The positive, negative, and overall agreement between the IMMULITE 2000 CMV IgM assay and the VIDAS CMV IgM assay (predicate assay) were determined. The assay demonstrated excellent reproducibility with a total CV of less than 10%. The positive, negative, and overall agreement between the IMMULITE 2000 assay and the VIDAS assay were > 95% for the method comparison samples. Among potentially cross-reactive samples, the overall agreement between the two assays was 96%. Similarly, among the immunocompromised and pregnant subjects, the overall agreement was ~96% and ~97%, respectively. The IMMULITE 2000 CMV IgM assay demonstrated excellent reproducibility, minimal cross-reactivity, and performance comparable to that of the VIDAS CMV IgM assay. It can aid in the diagnosis of acute CMV or recent CMV infection by qualitatively detecting the CMV IgM antibodies in human serum or plasma.

In Cuba, previous reports have shown an increase of epidemic KS, reaching a total of 120 cases by the end of 2007, despite the use of HAART. To evaluate and compare the role of human herpes virus 8 (HHV-8) viral loads in different compartments of AIDS-related Kaposi's sarcoma (AIDS-KS) patients real-time polymerase chain reaction (RT-PCR) was used to determine the genome copy number of HHV-8 in plasma, saliva, tissue and peripheral blood mononuclear cells (PBMC) of 49 AIDS-KS patients. Overall, 98% of AIDS-KS patients harbored detectable HHV-8. HHV-8 could be detected in 91.6% of KS tissue lesions showing the highest viral load (median log = 3.14 copies/100 ng DNA) followed by saliva and PBMC which were positive in 78%, and 69.2%; respectively. In contrast, HHV-8 was detected in only 37% of plasma samples, which also showed lower viral loads. Men who had sex with men (MSM) were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tissues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). These results emphasize the systemic nature of HHV-8-infection and demonstrate the possible role of saliva in HHV-8 transmission among MSM.

The presence of human cytomegalovirus (HCMV) in male genital tract suggests its vertical transmission with spermatozoa and the development of a potentially dangerous fetal infection. The objective of the present study was to evaluate the possibility of intracellular HCMV localization in male germ cells and to examine the effect of the virus on human spermatogenesis. Semen samples from 91 infertile and 47 fertile men were analyzed. HCMV was detected by real time PCR, rapid culture method and PCR in situ. Human testis organotypic culture and quantitative karyological analysis were used to investigate viral effects on spermatogenesis. Localization of HCMV in immature germ cells and spermatozoa was studied by immunostaining with monoclonal antibodies and ultrastructural analysis of infected organotypic culture. Viral DNA was detected in 12.3% samples of motile spermatozoa, while infectious activity only in 2.9% infertile and fertile men without statistically significant intergroup difference. According to PCR in situ, the mean percentage of infected cell in both groups was 1.5% (0.25%-15%), which can serve as a criterion for evaluating the risk of HCMV transmission. In HCMV-infected organotypic culture viral antigens were identified in spermatides on day 4, in spermatogonia and spermatocytes on day 8, and in spermatozoa on day 14. Empty and full capsides and virions were visualized in germ cells by electron microscopy. The number of cells before introduction in culture was taken for 100%. On day 14 infected culture contained 36.8% spermatogonia, 18.7% spermatocytes, 27.6% round spermatides and 42.5% elongated spermatides; in comparison with 82.2%, 51.5%, 70.4% and 65.7% in uninfected culture, respectively (all p < 0.05). There were no changes in the number and viability of spermatozoa. HCMV was detected in male germ cells, both in sperm samples and in testis organotypic culture. The virus may infect immature germ cells which develop to mature HCMV-carrying spermatozoa. A considerable decrease in the number of immature germ cells indicates that HCMV produces a direct gametotoxic effect and can contribute to male infertility.

Cytomegalovirus (CMV) infection has been associated with accelerated transplant vasculopathy. In this study, we assessed the effects of acute rat CMV (RCMV) infection on vessel remodeling in transplant vasculopathy, focusing on allograft morphology, inflammation and contribution of adventitial cells to intimal hyperplasia. Infrarenal aorta was locally infected with RCMV and transplanted from female F344 rats to male Lewis rats. Graft samples were collected 2 and 8 weeks after transplantation and analyzed for intimal hyperplasia, collagen degradation and inflammation. Transplantation of aorta followed by transplantation of RCMV infected and labeled isogenic adventitia were performed to study migration of adventitial cells towards the intima. Intimal hyperplasia was increased threefold in infected allografts. RCMV induced apoptosis in the media, expression of matrix metalloproteinase 2, and decreased collagen deposits. Macrophage infiltration was increased in the infected allografts and resulted in increased production of MCP-1. RCMV-infected macrophages were observed in the adventitia and intima. Cells derived from infected adventitia migrated towards the intima of the allograft. RCMV enhances infiltration of macrophages to the allografts, and thereby increases MCP-1 production and inflammation, followed by recruitment of adventitial cells to the intima and accelerated intimal hyperplasia.

Genetic mutant organisms pervade all areas of Biology. Early on, herpesviruses (HV) were found to be amenable to genetic analysis using homologous recombination techniques in eukaryotic cells. More recently, HV genomes cloned onto a bacterial artificial chromosome (BAC) have become available. HV BACs can be easily modified in E.coli and reintroduced in eukaryotic cells to produce infectious viruses. Mutants derived from HV BACs have been used both to understand the functions of all types of genetic elements present on the virus genome, but also to generate mutants with potentially medically relevant properties such as preventative vaccines. Here we retrace the development of the BAC technology applied to the Epstein-Barr virus (EBV) and review the strategies available for the construction of mutants. We expand on the appropriate controls required for proper use of the EBV BACs, and on the technical hurdles researchers face in working with these recombinants. We then discuss how further technological developments might successfully overcome these difficulties. Finally, we catalog the EBV BAC mutants that are currently available and illustrate their contributions to the field using a few representative examples.

Therapeutic vaccines can be designed to enhance existing T cell memory populations for increased protection against re-infection. In the case of herpes simplex virus type 1, recurrent disease results from reactivation of latent virus in sensory ganglia, which is controlled in part by a ganglia-resident HSV-specific memory CD8+ T cell population. Thus, an important goal of a therapeutic HSV-1 vaccine would be to enhance this population. HSV-1-infected mice were treated with TAK-779 to block CCR5- and CXCR3-mediated CD8+ T cell migration during both acute and latent infections. Additionally, HSV-1-specific CD8+ T cells were transferred into HSV-1 latently infected mice to mimic the effect of a therapeutic vaccine, and their migration into trigeminal ganglia (TG) was traced during steady-state latency, or during recovery of the TG-resident memory CD8+ T cell population following stress-, and corticosterone-induced depletion and HSV-1 reactivation from latency. Bromodeoxy uridine (BrdU) incorporation measured cell proliferation in vivo. TAK-779 treatment during acute HSV-1 infection reduced the number of infiltrating CD8+ T cells but did not alter the number of viral genome copies. TAK-779 treatment during HSV latency did not affect the size of the TG-resident memory CD8+ T cell population. Transferred HSV-specific CD8+ T cells failed to access latently infected TG during steady-state latency, or during recovery of the TG resident HSV-specific CD8+ T cell population following exposure of latently infected mice to stress and corticosterone. Recovery of the HSV-specific CD8+ T cell population after stress and corticosterone treatment occurred with homeostatic levels of cell division and did not require CD4+ T cell help. Our findings are consistent with the notion that the CD8+ T cells in latently infected TG are a tissue-resident memory (Trm) population that is maintained without replenishment from the periphery, and that when this population is disrupted, it recovers without proliferation or detectable recruitment of HSV-specific CD8+ T cells from the blood. The compartmentalization of the HSV-specific CD8+ memory T cell population in latently infected TG will complicate the design of therapeutic vaccines.