Human herpesvirus 8 load and progression of AIDS-related Kaposi sarcoma lesions

Introduction—Human herpesvirus 8 (HHV8) is necessary for Kaposi sarcoma (KS) to develop, but whether peripheral blood viral load is a marker of KS burden (total number of KS lesions), KS progression (the rate of eruption of new KS lesions), or both is unclear. We investigated these relationships in persons with AIDS. Methods—Newly diagnosed patients with AIDS-related KS attending Mulago Hospital, in Kampala, Uganda, were assessed for KS burden and progression by questionnaire and medical examination. Venous blood samples were taken for HHV8 load measurements by PCR. Associations were examined with odds ratio (OR) and 95% confidence intervals (CI) from logistic regression models and with t-tests. Results—Among 74 patients (59% men), median age was 34.5 years (interquartile range [IQR], 28.5-41). HHV8 DNA was detected in 93% and quantified in 77% patients. Median virus load was 3.8 logs10/106 peripheral blood cells (IQR 3.4-5.0) and was higher in men than women (4.4 vs. 3.8 logs; p=0.04), in patients with faster (>20 lesions per year) than slower rate of KS lesion eruption (4.5 vs. 3.6 logs; p<0.001), and higher, but not significantly, among patients with more (>median [20] KS lesions) than fewer KS lesions (4.4 vs. 4.0 logs; p=0.16). HHV8 load was unrelated to CD4 lymphocyte count (p=0.23). Conclusions—We show significant association of HHV8 load in peripheral blood with rate of eruption of KS lesions, but not with total lesion count. Our results suggest that viral load increases concurrently with development of new KS lesions.

Microarrays in the Diagnosis of Human Herpesvirus infections

Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections.

Human herpesvirus-6 infection in liver transplantation

Rejection and infections are the two most common complications after liver transplantation. Human herpesvirus-6 (HHV-6) belongs to the betaherpesviruses, together with its close relatives cytomegalovirus (CMV) and human herpesvirus-7 (HHV-7). The impact of CMV in liver transplantation is well characterized, but the roles of the other two betaherpesviruses have been acknowledged only recently. Although, HHV-6 reactivation after transplantation is usually asymptomatic, the virus may infect the liver transplant, cause an intragraft lymphocyte dominated inflammatory reaction and graft dysfunction. HHV-6 is also suggested to be associated with liver allograft rejection but the mechanisms are unclear. The aim of this study was to investigate the intragraft immunological processes associated with HHV-6, the involvement of HHV-6 in acute liver failure (ALF) and the hepatic HHV-6 infection of the same patients after transplantation. In addition, the occurrence of HHV-6 and HHV-7 was investigated in liver transplant patients with symptomatic CMV infection. HHV-6 infection of the liver graft was associated with portal lymphocyte infiltration and with a significant increase of adhesion molecules (ICAM-1 and VCAM-1) and the number of cells expressing their ligand molecules (LFA-1, VLA-4) and class II antigens. HHV-6 infection was associated with significant immunological changes, but the immune response was limited to lymphocyte infiltration and the adhesion molecule level. However, one third of these patients developed chronic rejection during the follow-up. Of the patients with ALF of unknown origin, most patients demonstrated HHV-6 antigens in the liver, whereas the opposite was seen in ALF patients with a known disease. After transplantation, HHV-6 recurrence was found in the liver transplant in half of these patients with pre-transplant HHV-6 infection of the liver, whereas no post-transplant HHV-6 infection of the liver was seen in patients without pre-transplant HHV-6. Our studies further demonstrated that both HHV-6 and HHV-7 antigenemia often appeared in association with CMV disease in liver transplant patients. The time-related occurrence of the viruses differed, as HHV-6 appeared early after transplantation and regularly preceded CMV whereas HHV-7 often appeared concurrently with CMV. In conclusion, these results indicate that all three betaherpesviruses are common after liver transplantation, often associated with each other. The immunological events caused by HHV-6 in the liver transplant may be involved in, or trigger mechanisms of allograft rejection. In addition, HHV-6 could be one of the causes of ALF, and pre-transplant HHV-6 infection in ALF patients is a risk factor for post-transplant HHV-6 infection of the graft. These results strongly support the clinical significance of HHV-6 in liver transplantation. Even though the reactivation is usually asymptomatic, in some individuals HHV-6 infection may lead to severe manifestations, such as liver failure or in transplant patients, graft dysfunction and rejection.

Patterns of Antibodies Against Latent and Lytic Antigens of Human Herpesvirus 8 in an Endemic Population and Patients With Kaposi`s Sarcoma in Mozambique

The patterns of antibodies against latent and lytic antigens of human herpesvirus 8 (HHV-8) were assessed using immunofluorescence assays of samples from 155 persons seropositive for HHV-8 seen at public health centers and 24 patients with Kaposi`s sarcoma (KS) from Mozambique. Of the 155 persons without KS, 48(31%) had antibodies against latent antigens only, 29 (18.7%) had antibodies against lytic antigens only, and 78 (50.3%) had antibodies against both types of antigen. The HHV-8 antibody titer tended to increase with age until age 40, after which it began to decrease. High titers of antibodies against latent and lytic antigens of HHV-8 were detected mostly in persons co-infected with HIV, and these increased titers could have a predictive value. All patients with KS except four patients who were seronegative for HHV-8 had elevated titers of HHV-8 antibodies, predominantly against latent antigens. The data suggest the potential for an increase in the development of KS in this endemic area for HHV-8. J. Med. Virol. 82:1576-1581, 2010. (C) 2010 Wiley-Liss, Inc.

Seroprevalence of Human Herpesvirus 8 Infection in Individuals From Health Care Centers in Mozambique: Potential for Endemic and Epidemic Kaposi`s Sarcoma

Human herpesvirus 8 (HHV-8) infection is common in sub-Saharan Africa, but its prevalence in Mozambique is unknown. The seroprevalence of HHV-8 in a cohort of individuals seen at public health centers in Northern (n = 208), Central (n = 226), or Southern (n = 318) Mozambique was examined. All individuals were interviewed to obtain socioeconomic, demographic and clinical data and were tested for serum anti-HHV-8 antibodies using an immunofluorescence assay. The overall frequency of HHV-8 antibodies was 21.4% and, in spite of the diversity of epidemiological characteristics of the tested individuals, did not differ significantly among regions: 18.7%, 24.3% and 21.4% in the North, Center, and South, respectively (chi(2), 2.37; P = 0.305). The variables that were associated significantly with the presence of HHV-8 antibodies were gender, age, level of education, number of siblings and HIV serostatus, but these differed across the regions. In the North, although tested individuals lived under poor socioeconomic conditions, no association between HHV-8 infection and household variables was detected, with the exception of the number of siblings (P = 0.042). In the Central region, HHV-8 infection was associated with gender (P = 0.010), the number of household members (P = 0.031), and the place of attendance (P = 0.021). In the South, HHV-8 infection was associated with the number of siblings (P = 0.023) and HIV status (P = 0.002). The overall prevalence of HHV-8 seropositivity increased with age. These results demonstrate that Mozambique is another country in Africa with endemic HHV-8 infection, and, because of the AIDS epidemic, continued access to antiretroviral treatment is necessary to avert an outbreak of AIDS-Kaposi`s sarcoma. J. Med. Virol. 82:1216-1223, 2010. (C) 2010 Wiley-Liss, Inc.

A Comparative Study of the Frequency of Antibody and Titers Against Human Herpesvirus 8 Latent and Lytic Antigens in ""At-Risk"" Individuals and Among Patients With Kaposi`s Sarcoma

Differences in the prevalence of human herpesvirus 8 (HHV-8) and Kaposi`s sarcoma (KS) have been described, depending on the study population and their geographic origin. A cross-sectional study aimed at detecting the frequency and titers of antibodies against HHV-8 latent and lytic antigens in serum samples from individuals with different risk-factors for HHV-8 infection, as well as predictive marker identification in patients with KS, was conducted. Serum samples were collected from seven groups of individuals: 75 patients with AIDS-KS, 5 with classic KS, 16 with African KS, 495 with HIV/AIDS, 805 patients with chronic kidney disease, 683 handicapped individuals, and 757 health care workers. Samples were evaluated for the presence and titers of HHV-8-specific antibodies to latent and lytic antigens using ""in house"" immunofluorescence assays. The results were analyzed by the Chi-square, Fisher`s exact test, Kruskal-Wallis and/or Mann-Whitney U-tests. The frequencies of HHV-8 antibodies were as follows: 87.5-100% in patients with KS, 20.4% in patients with HIV/AIDS, 18% in patients with chronic kidney disease, 1.6% in handicapped individuals, and 1.1% in health care workers. A greater number of samples were antibody positive to lytic antigens. Elevated titers of antibodies to latent and lytic antigens, mostly among patients with KS, were detected. Using established serological assays, different ""at-risk"" populations for HHV-8 infection/disease were detected in this geographic area, confirming HIV/AIDS and identifying patients with chronic kidney disease as high-risk groups. It is suggested that a longitudinal evaluation of antibody titers in patients with chronic kidney disease be undertaken to confirm their predictive value in the development of KS. J. Med. Virol. 81: 1292-1297, 2009. (C) 2009 Wiley-Liss, Inc.

Transmission of Human Herpesvirus Type 8 Infection Within Families in American Indigenous Populations From the Brazilian Amazon

Background. The intrafamilial dynamics of endemic infection with human herpesvirus type 8 (HHV-8) in Amerindian populations is unknown. Methods. Serum samples were obtained from 517 Amerindians and tested for HHV-8 anti-latent nuclear antigen (anti-LANA) and antilytic antibodies by immunofluorescence assays. Logistic regression and mixed logistic models were used to estimate the odds of being HHV-8 seropositive among intrafamilial pairs. Results. HHV-8 seroprevalence by either assay was 75.4% (95% confidence interval [CI]: 71.5%-79.1%), and it was age-dependent (P-trend<.001). Familial dependence in HHV-8 seroprevalence by either assay was found between mother-offspring (odds ratio [OR], 5.44; 95% CI: 1.62-18.28) and siblings aged >= 10 years (OR 4.42, 95% CI: 1.70-11.45) or siblings in close age range (<5 years difference) (OR 3.37, 95% CI: 1.21-9.40), or in families with large (>4) number of siblings (OR, 3.20, 95% CI: 1.33-7.67). In separate analyses by serological assay, there was strong dependence in mother-offspring (OR 8.94, 95% CI: 2.94-27.23) and sibling pairs aged >= 10 years (OR, 11.91, 95% CI: 2.23-63.64) measured by LANA but not lytic antibodies. Conclusions. This pattern of familial dependence suggests that, in this endemic population, HHV-8 transmission mainly occurs from mother to offspring and between close siblings during early childhood, probably via saliva. The mother to offspring dependence was derived chiefly from anti-LANA antibodies.

Infection with human herpesvirus 8 and transplant-associated gammopathy

BACKGROUND: The role of human herpesvirus (HHV)-8 in the pathogenesis of multiple myeloma and its pre-malignant state of monoclonal gammopathy is unclear. HHV-8 is transmitted by organ transplantation, representing a unique model with which to investigate primary HHV-8 infection. METHODS: The authors studied the incidence of clonal gammopathy in renal transplant recipients and correlated it with previous and recent HHV-8 infection. RESULTS: Clonal gammopathy was observed in 31 of 162 (19%) HHV-8-seronegative patients, in 5 of 17 (29%) HHV-8-seropositive patients, and in 9 of 24 (38%) HHV-8 seroconverters within 5 years after transplantation. Gammopathy was often transient, and no progression to myeloma was observed. Two patients with persistent gammopathy developed B-cell lymphoma. In a logistic regression model, HHV-8 serostatus of the graft recipient was significantly associated with subsequent development of gammopathy, with a relative risk (RR) of 1.9 and a 95% confidence interval (CI) of 0.5 to 6.4 for an HHV-8-seropositive recipient and an RR of 2.9 and a 95% CI of 1.01 to 8.0 for seroconverters as compared with baseline (HHV-8 seronegative). Other significant variables were cytomegalovirus (CMV) serostatus and the intensity of immunosuppression (RR of 10.4 and 95% CI of 2.6-41.7 for a CMV-negative recipient with a CMV-positive donor vs. a CMV-negative recipient with a CMV-negative donor and RR of 17.6 and 95% CI of 2.0-150.8 if OKT3 was used vs. no use of antilymphocytic substances). CONCLUSIONS: Transplant recipients with HHV-8 infection are more likely to develop clonal gammopathy. However, this risk is much lower than the risk conferred by CMV infection and antilymphocytic therapy, arguing against a major role of HHV-8 infection in the pathogenesis of clonal plasma cell proliferation.

Is human herpesvirus 8 infection more common in men than in women? Systematic review and meta-analysis.

All forms of Kaposi sarcoma (KS) are more common in men than in women. It is unknown if this is due to a higher prevalence of human herpesvirus 8 (HHV-8), the underlying cause of KS, in men compared to women. We did a systematic review and meta-analysis to examine the association between HHV-8 seropositivity and gender in the general population. Studies in selected populations like for example, blood donors, hospital patients, and men who have sex with men were excluded. We searched Medline and Embase from January 1994 to February 2015. We included observational studies that recruited participants from the general population and reported HHV-8 seroprevalence for men and women or boys and girls. We used random-effects meta-analysis to pool odds ratios (OR) of the association between HHV-8 and gender. We used meta-regression to identify effect modifiers, including age, geographical region and type of HHV-8 antibody test. We included 22 studies, with 36,175 participants. Men from sub-Saharan Africa (SSA) (OR 1.21, 95% confidence interval [CI] 1.09-1.34), but not men from elsewhere (OR 0.94, 95% CI 0.83-1.06), were more likely to be HHV-8 seropositive than women (p value for interaction=0.010). There was no difference in HHV-8 seroprevalence between boys and girls from SSA (OR 0.90, 95% CI 0.72-1.13). The type of HHV-8 assay did not affect the overall results. A higher HHV-8 seroprevalence in men than women in SSA may partially explain why men have higher KS risk in this region. This article is protected by copyright. All rights reserved.

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

Human Kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Antibodies against human herpesvirus 8 in black South African patients with cancer

Background Infection with human herpesvirus 8 (HHV-8) has been consistently linked to Kaposi's sarcoma, but its mode of transmission, association with other cancers, and interaction with the human immunodeficiency virus type 1 (HIV-1) are largely unknown. Methods Between January 1992 and December 1997, we interviewed 3591 black patients with cancer in Johannesburg and Soweto, South Africa. Blood was tested for antibodies against HIV-1 and HHV-8 in 3344 of the patients. Antibodies against HHV-8 were detected with an indirect immunofluorescence assay. The intensity of the fluorescent signal correlated well with the titers of antibodies (P<0.001). The relations among the presence of anti–HHV-8 antibodies, sociodemographic and behavioral factors, type of cancer, and the presence or absence of coexistent HIV-1 infection were examined with the use of unconditional logistic-regression models. Results Among the 3293 subjects with cancers other than Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 32 percent, which did not differ significantly from the standardized seroprevalence among black blood donors. Among these 3293 patients, the prevalence of antibodies against HHV-8 increased with increasing age (P<0.001) and an increasing number of sexual partners (P=0.05) and decreased with increasing years of education (P=0.007); it was not strongly associated with HIV-1 infection. Anti–HHV-8 antibodies were more frequent among black than white blood donors (P<0.001). Among the 51 patients with Kaposi's sarcoma, the standardized seroprevalence of antibodies against HHV-8 was 83 percent, significantly higher than the prevalence among those without Kaposi's sarcoma (P<0.001). For 16 other specific types of cancer, including multiple myeloma (108 cases) and prostate cancer (202 cases), the variation in the standardized seroprevalence of antibodies against HHV-8 was not remarkable. At a given intensity of fluorescence of anti–HHV-8 antibodies, Kaposi's sarcoma was more frequent among HIV-1–positive patients than among those who were HIV-1–negative (P<0.001). Conclusions Among black patients with cancer in South Africa, the seroprevalence of anti–HHV-8 antibodies is high and is specifically associated with Kaposi's sarcoma, particularly at high titers.

Sex differences in the human connectome: 4-Tesla high angular resolution diffusion imaging (HARDI) tractography in 234 young adult twins

Cortical connectivity is associated with cognitive and behavioral traits that are thought to vary between sexes. Using high-angular resolution diffusion imaging at 4 Tesla, we scanned 234 young adult twins and siblings (mean age: 23.4 2.0 SD years) with 94 diffusion-encoding directions. We applied a novel Hough transform method to extract fiber tracts throughout the entire brain, based on fields of constant solid angle orientation distribution functions (ODFs). Cortical surfaces were generated from each subject's 3D T1-weighted structural MRI scan, and tracts were aligned to the anatomy. Network analysis revealed the proportions of fibers interconnecting 5 key subregions of the frontal cortex, including connections between hemispheres. We found significant sex differences (147 women/87 men) in the proportions of fibers connecting contralateral superior frontal cortices. Interhemispheric connectivity was greater in women, in line with long-standing theories of hemispheric specialization. These findings may be relevant for ongoing studies of the human connectome.

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.