Human herpesvirus 8 – A novel human pathogen

In 1994, Chang and Moore reported on the latest of the gammaherpesviruses to infect humans, human herpesvirus 8 (HHV-8) [1]. This novel herpesvirus has and continues to present challenges to define its scope of involvement in human disease. In this review, aspects of HHV-8 infection are discussed, such as, the human immune response, viral pathogenesis and transmission, viral disease entities, and the virus's epidemiology with an emphasis on HHV-8 diagnostics.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

High Human Herpesvirus 8 (HHV-8) Prevalence,Clinical Correlates and High Incidence among Recently HIV-1-Infected Subjects in Sao Paulo,Brazil

Background

Human herpesvirus 8 (HHV-8) is the etiological agent for Kaposi Sarcoma, which occurs especially in HIV-infected subjects. HHV-8 infection and its clinical correlates have not been well characterized in recently HIV-1-infected subjects, especially men who have sex with men (MSM).

Methodology/ Principal Findings

We assessed the HHV-8 seroprevalence, clinical correlates, and incidence after one year of follow-up in a cohort of 228 recently HIV-1-infected individuals, of whom 83.6% were MSM, using indirect immunofluorescence assay. The prevalence of HHV-8 infection at the time of cohort enrollment was 25.9% (59/228). In the univariate model, there were significant associations with male gender, black ethnicity, MSM practice, and previous hepatitis B virus and syphilis infections. In the multivariate model we could still demonstrate association with MSM, hepatitis B, and black ethnicity. No differences in mean CD4+ cell counts or HIV viral load according to HHV-8 status were found. In terms of incidence, there were 23/127 (18.1%) seroconversions in the cohort after 1 year.

Conclusions

HHV-8 is highly prevalent among recently HIV-1-infected subjects. Correlations with other sexually transmitted infections suggest common transmission routes.     

DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.     

Comparative prevalence of plasma human herpesvirus 8 DNA in sexual contact and intravenous injection routes of HIV transmission

Detection of plasma human herpesvirus (HHV)-8 DNA correlates with antibodies to lytic HHV-8 antigens, being predictive of Kaposi's sarcoma in HIV-infected patients. We show that the prevalence of plasma HHV-8 DNA was 10.6% for HIV infection through sexual contact and 7.1% for HIV infection through intravenous injection. In addition, the prevalence of plasma HHV-8 DNA was significantly associated with male gender (9.4%) and HIV viral load below 1000 copies mL(-1) (12.1%), but not age or CD4 cell count in HIV-infected patients. The study suggested that detection of plasma HHV-8 DNA could be important for monitoring replicating HHV-8 in HIV-infected patients, and may have use as a marker for the diagnosis of HHV-8 infection in blood-borne transmission.     

Seroprevalence of herpes simplex virus type 2 in adult HIV-infected patients and blood donors in Croatia

The present study estimates herpes simplex virus type 2 (HSV-2) seroprevalence and evaluates its association with age, sex, human herpesvirus type 8 (HHV-8) and human immunodeficiency virus (HIV) among adults in Croatia. A cross-sectional survey included 166 HIV-infected patients and 219 blood donors. Antibodies against HSV-2 were determined by enzyme immunoassays based on gG2 recombinant glycoprotein. HSV-2 seroprevalence was 45.8% in HIV-infected patients and 8.7% in blood donors (p < 0.0001; OR 8.8; 95% CI 5.05-15.49). Independent predictors of HSV-2 seropositivity were HIV infection (OR 11.0; 95% CI 5.93-20.41), female gender (OR 2.28; 95% CI 1.22-4.26), older age (OR 3.93; 95% CI 2.74-7.11), and HHV-8 seropositivity (OR 2.72; 95% CI 1.09-6.75). Understanding the epidemiology of HSV-2 is a critical first step in designing interventions to decrease HSV-2 and HIV transmission. The association of HSV-2 with HIV infection and HHV-8 antibodies suggests a similar transmission route.     

Human herpesvirus 8 infects and replicates in primary cultures of activated B lymphocytes through DC-SIGN

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.     

人类8型疱疹病毒的研究进展

人类8型疱疹病毒（human herpesvirus-8,HHV-8）又称卡波氏肉瘤相关疱疹病毒（Kapo- si's sarcoma- associated herpesvirus,KSHV）,是一种新的肿瘤病毒,目前被认为是卡波氏肉瘤（Kaposi's sarcoma,KS）致病因子,并且与primary effusion lymphoma （PEL）和multicentric Castleman's disease（MCD）相关。该病毒编码许多蛋白,包括潜伏感染相关蛋白,裂解感染相关蛋白和HHV-8特有基因表达蛋白,在KS和HHV-8相关疾病的发病中起到关键作用。     

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.     

Locatization of human herpesvirus type 8 in human sperms by in situ PCR

SummaryObjectives: Defining the mechanism of infection with human herpesvirus-8 (HHV-8) or Kaposi’s sarcoma associated herpesvirus (KSHV) is an important clinical issue. HHV-8 has been linked to Kaposi’s sarcoma (KS) development in HIV-1-infected individuals, and KS develops in 40% of those infected with both viruses. A series of epidemiological data suggest that sexual transmission is one of the routes of transmission for HHV-8. In our studies, we sought to assess the cellular reservoirs of HHV-8 DNA in the semen of HIV-1-infected men and the potential role of HHV-8 infected spermatozoa in horizontal transmission.Design and methods: A nested polymerase chain reaction (PCR), in situ PCR (ISPCR) and a sodium iodide (NaI) DNA isolation technique that extracts both nuclear and episomal DNA were utilized to amplify specific genes in vitro and within intact cells to evaluate the types of seminal cells infected with HHV-8 in HIV-1-infected and uninfected men.Results HHV-8 was present in the spermatozoa and mononuclear cells of the semen in 64 of 73 (88%) HIV-1 infected individuals. Both the sperms as well as the mononuclear cells of the semen specimens of HIV-1 infected men were found to be infected with HHV-8. Multiplex ISPCR revealed that a significantly higher percentage of semen cells were infected with HHV-8 than HIV-1 (p>0.001). Rare (less than one in a 100,000) sperm cells were co-infected with both viruses. A co-culture of HHV-8 infected sperm with uninfected 293 or Sup-T1 cell lines resulted in an abortive infection of these cells with HHV-8. DNA isolation by NaI yielded 73% of the positive sperm, whereas the standard phenol/chloroform method resulted in significantly lower positives (45%) from the same specimens.Conclusions: Design and methods: Our data strongly suggest a potential sexual/horizontal route of transmission of HHV-8, via the HHV-8 infected sperm and other semen cells, where a large percentage of HIV-1 infected men’s sperm and other semen cells are infected with HHV-8. Co-culture studies have further supported the observations that HHV-8 in the sperm cells is infectious and capable of transmission of the virus to uninfected cells.     

Molecular biology and pathogenesis of hepatitis E virus

The hepatitis E virus (HEV) is a small RNA virus and the etiological agent for hepatitis E, a form of acute viral hepatitis. The virus has a feco-oral transmission cycle and is transmitted through environmental contamination, mainly through drinking water. Recent studies on the isolation of HEV-like viruses from animal species also suggest zoonotic transfer of the virus. The absence of small animal models of infection and efficient cell culture systems has precluded virological studies on the replication cycle and pathogenesis of HEV. A vaccine against HEV has undergone successful clinical testing and diagnostic tests are available. This review describes HEV epidemiology, clinical presentation, pathogenesis, molecular virology and the host response to HEV infection. The focus is on published literature in the past decade. Equal contribution     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Effect of acute Plasmodium falciparum malaria on reactivation and shedding of the eight human herpes viruses

Human herpes viruses (HHVs) are widely distributed pathogens. In immuno-competent individuals their clinical outcomes are generally benign but in immuno-compromised hosts, primary infection or extensive viral reactivation can lead to critical diseases. Plasmodium falciparum malaria profoundly affects the host immune system. In this retrospective study, we evaluated the direct effect of acute P. falciparum infection on reactivation and shedding of all known human herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8). We monitored their presence by real time PCR in plasma and saliva of Ugandan children with malaria at the day of admission to the hospital (day-0) and 14 days later (after treatment), or in children with mild infections unrelated to malaria. For each child screened in this study, at least one type of HHV was detected in the saliva. HHV-7 and HHV-6 were detected in more than 70% of the samples and CMV in approximately half. HSV-1, HSV-2, VZV and HHV-8 were detected at lower frequency. During salivary shedding the highest mean viral load was observed for HSV-1 followed by EBV, HHV-7, HHV-6, CMV and HHV-8. After anti-malarial treatment the salivary HSV-1 levels were profoundly diminished or totally cleared. Similarly, four children with malaria had high levels of circulating EBV at day-0, levels that were cleared after anti-malarial treatment confirming the association between P. falciparum infection and EBV reactivation. This study shows that acute P. falciparum infection can contribute to EBV reactivation in the blood and HSV-1 reactivation in the oral cavity. Taken together our results call for further studies investigating the potential clinical implications of HHVs reactivation in children suffering from malaria.     

Herpesvirus: an underestimated virus

Herpes simplex virus (HSV) infections are common and widespread; nevertheless, their outcome can be of unpredictable prognosis in neonates and in immunosuppressed patients. Anti-HSV therapy is effective, but the emergence of drug-resistant strains or the drug toxicity that hamper the treatment is of great concern. Vaccine has not yet shown relevant benefit; therefore, palliative prophylactic measures have been adopted to prevent diseases. This short review proposes to present concisely the history of HSV, its taxonomy, physical structure, and replication and to explore the pathogenesis of the infection, clinical manifestations, laboratory diagnosis, treatment, prophylaxis and epidemiology of the diseases.     

Kaposi's sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-zeta-MEK-ERK signaling pathway in target cells early during infection: implications for infectivity

Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus alpha3 and beta1 complexes, and virus-binding studies suggest a role for alpha3beta1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-zeta (PKC-zeta) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble alpha3beta1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human alpha3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the alpha3beta1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-zeta, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-zeta, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8's infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.     

GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.     

Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one

Recent research has provided new insights on the epidemiology, pathology and immunology of canine leishmaniosis (CanL) and its genetic basis. The prevalence of infection in endemic areas is considerably higher than that of apparent clinical illness. In addition, infection spreads rapidly among dogs in the presence of optimal conditions for transmission. Infection involves a variety of granulomatous and harmful immune-mediated responses, and susceptibility to the disease is influenced by a complex genetic basis. These concepts will be instrumental for devising control programs. This review, the first in a series of two articles on CanL, presents an updated view on progress in elucidating the epidemiology and pathogenesis of this challenging disease, and the second part focuses on advances in diagnosis, treatment and prevention.     

Characterization of entry mechanisms of human herpesvirus 8 by using an Rta-dependent reporter cell line

To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.