More than 100 herpesviruses have been discovered, of which all are double-stranded DNA viruses that can establish latent infections in their respective vertebrate hosts; however, only eight regularly infect humans. The Herpesvirinea family is subdivided into three subfamilies: the Alpha-, Beta-, or Gammaherpesvirinea. This classification was created by the Herpesvirus Study Group of the International Committee on Taxonomy of Viruses using biological properties and it does not rely upon DNA sequence homology. However, researchers have been able to identify and appropriately characterize the viral subfamilies using DNA sequence analysis of the DNA polymerase gene; other investigators have been successful using the glycoprotein B gene 2.

The Alphaherpesvirinea are defined by variable cellular host range, shorter viral reproductive cycle, rapid growth in culture, high cytotoxic effects, and the ability to establish latency in sensory ganglia. In humans, these are termed herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV), and represent human herpesviruses 1, 2, and 3 2.

The Betaherpesvirinea have a more restricted host range with a longer reproductive viral cycle and slower growth in culture. Infected cells show cytomegalia (enlargement of the infected cells). Latency is established in secretory glands, lymphoreticular cells, and in tissues such as the kidneys among others. In humans, these are termed human cytomegalovirus (HCMV or herpesvirus 5), human herpesviruses 6A and 6B (HHV-6A and -6B), and human herpesvirus 7 (HHV-7). HHV-7 has also been called the roseolavirus, after the disease roseola infantum it causes in children 2.

The Gammaherpesvirinea have a host range that is found within organisms that are part of the Family or Order of the natural host. In vitro replication of the viruses occurs in lymphoblastoid cells, but some lytic infections occur in epithelial and fibroblasts for some viral species in this subfamily. Gammaherpesviruses are specific for either B or T cells with latent virus found in lymphoid tissues. Only two human Gammaherpesviruses are known, human herpesvirus 4, referred to as Epstein-Barr virus (EBV), and human herpesvirus 8, referred to as HHV-8 or Kaposi's sarcoma-associated herpesvirus (KSHV) 2. The gammaherpesviruses subfamily contains two genera (a classification of closely related viruses) that includes both the gamma-1 or Lymphocryptovirus (LCV) and the gamma-2 or Rhadinovirus (RDV) virus genera. EBV is the only LCV and HHV-8 is the only RDV discovered in humans. LCV is found only in primates but RDV can be found in both primates and subprimate mammals. RDV DNAs are more diverse across species and are found in a broader range of mammalian species. It is thought that RDVs evolved before LCVs 2.

HHV-8 has sequence homology and genetic structure that is close to another RDV, Herpesvirus saimiri (HVS) 3. HVS can cause fulminant T-cell lymphoma in its primate host and can immortalize infected T-cells 4. Rhadinaviruses can infect ungulates, mice, and rabbits and all share a particular genomic organization characterized by large flanking, highly repetitive DNA repeats of high G/C content 5.

The phenotypic architecture of the Herpesviridae family viruses characterizes these viruses. Customarily, herpesviruses have a central viral core that contains a linear double stranded DNA. This DNA is in the form of a torus, exemplified by a hole through the middle and the DNA is embedded in a proteinaceous spindle 6. The capsid is icosadeltahedral (16 surfaces) with 2-fold symmetry and a diameter of 100–120 nm that is partially dependent upon the thickness of the tegument. The capsid has 162 capsomeres. The three dimensional structure of the HHV-8 capsid was determined by cryo-electron microscopy (EM) and was found to be composed of 12 pentons, 150 hexons, and 320 triplexes arranged as expected in the icosadeltahedral lattice with 20 faces; the capsids are 125 nm in diameter 7. Transmission EM showed a bulls-eye appearance in the virions with electron dense cores and amorphous teguments surrounding the viral core 8. Interestingly, these structural characteristics were seen in endemic KS lesions as early as 1984, but were not recognized at that time as the possible etiology of the disease 9.

The herpesvirus tegument, an amorphorous proteinaceous material that under EM lacks distinctive features, is found between the capsid and the envelope; it can be asymmetric in distribution. Thickness of the tegument is variable dependent upon its location in the cell and varies between different herpesviruses 10.

The herpesvirus envelope contains viral glycoprotein protrusions on the surface of the virus 2. As shown by EM there is a trilaminar appearance 11 derived from the cellular membranes 12 and contains some lipid 13. Glycoproteins protrude from the envelope and are more numerous and shorter than those found on other viruses. The presence of the envelope can influence the size measurement of the virus under EM conditions 2.

There are six defined DNA genomic sequence arrangements for viruses in the Herpesviridae family. Of the human herpesviruses, EBV and HHV-8 are in class C. In this grouping, the number of direct terminal repeats are smaller than for other herpesviruses and there are other repeats found within the genome itself that subdivide the genome into unique stretches 2. All known herpesviruses have capsid packaging signals at their termini 14.

The majority of herpes genes contain upstream promoter and regulatory sequences, an initiation site followed by a 5' nontranslated leader sequence, the open reading frame (Orf) itself, some 3' nontranslated sequences, and finally, a polyadenylation signal. There are exceptions to this format because initiation from an internal in-frame methionine has been reported 15.

Gene overlaps are common, whereby the promoter sequences of antisense strand (3') genes are located in the coding region of sense strand (5') genes; Orfs can be antisense to one another. Proteins can be embedded within larger coding sequences and yet have different functions. Most genes are not spliced and therefore are without introns and sequences for noncoding RNAs are present 2.

Herpesviruses code for genes that code for proteins involved in establishment of latency, production of DNA, and structural proteins for viral replication, nucleic acid packaging, viral entry, capsid envelopment, for the blocking or modifying host immune defenses, and transitions from latency to lytic growth. Although all herpesviruses establish latency, some (e.g., HSV) do not absolutely require latent protein expression to remain in latency, unlike others (e.g., EBV and HHV-8). Herpesviruses can alter their environment by affecting host cell protein synthesis, host cell DNA replication, immortalizing the host cell, and the host's immune responses (e.g., blocking apoptosis, cell surface MHC I expression, modulation of the interferon pathway) 2.

Gene expression is occurs in two major stages: latency and lytic growth. In the latent phase, there can be replication of circular episomal DNA, and latency typically involves the expression of only a few latently expressed genes. Generally, most host cells infected by herpesviruses exist in a latent phase. When KS tissue or BCBL-1 HHV-8 infected cultured cells are analyzed 8, the vast majority of the infected cells are infected with latent HHV-8 virus. Only a small percent of the cells (≤ 1%) appear to be undergoing lytic replication in a latently infected cell line 16.

The herpesvirus lytic replicative phase can itself be divided into four stages:

1. α or immediate early (IE), which requires no prior viral protein synthesis. In the IE stage, genes involved in trans-activating transcription from other viral genes are expressed.

2. β or early genes (E), whose expression is independent of viral DNA synthesis.

3. Following the E phase, γ1 or partial late genes are expressed in concert with the beginning of viral DNA synthesis.

4. γ2 or late genes, where viral protein expression is totally dependent upon synthesis of viral DNA and where the expression of virion structural genes encoding for capsid proteins and envelope glycoproteins occurs.

HHV-8 shares four main biological properties with other herpesviruses:

1. A broad array of enzymes involved in nucleic acid metabolism, DNA synthesis, and protein processing.

2. DNA synthesis and capsid formation occur in the nucleus of the host cell and the viral capsid is enveloped at the nuclear membrane.

3. Production of infectious progeny virus in the lytic phase can kill the host cell.

4. The virus can attain a latent state in the host cell with closed circular episomes and a minimal amount of gene expression. Latent genomes, however, can become lytic with the proper stimulation using chemical agents such as sodium butyrate 2.

Several human host cells are permissive for HHV-8 infection. Two prototype cells are the B-cells of the body-cavity-based lymphoma (BCBL) or pleural effusion lymphoma (PEL) 23 and the spindle cells characteristic of Kaposi's sarcoma (KS) 24. Renne et al. 25 surveyed 38 mammalian cell lines or cell types and was only able to detect by RT-PCR the presence of infectivity from BCBL-1 derived virions in 11 of the 38. However, at least one cell type from lymphoid, endothelial, epithelial, fibroblastoid, and cancer cell types was permissive for infection. The 293 human kidney epithelial cell line was most susceptible in that study 25. Natural cellular reservoirs for HHV-8 are CD19+ B-cells 26. Natural infection in other cell types have been reported for endothelium 27, monocytes 28, prostate glandular epithelium 29, dorsal root sensory ganglion cells 30, and spindle cells of KS tumors 27.

Like other rhadinoviruses, HHV-8 might only be pathogenic when other cofactors are involved, such as concurrent infection with HIV or in an immunocompromised host. In the natural healthy host, the virus is relatively benign 5, however, currently, there is no known host other than humans.

LCV (EBV) and RDV (HHV-8) genomes are more closely related to each other than to the alpha- and betaherpesviruses 18. HHV-8 does not immortalize B-cells in vitro, as does EBV. HHV-8 has similar large reiterations of the TR as found with EBV but lack EBV's long internal repeats. HHV-8 possesses genes coding for dihydrofolate reductase (DHFR), interferon regulatory factor (IRF), G-protein coupled receptor (GPCR), chemokine analogs, and cyclin-D that are absent from the EBV genome 19. Fifty-four of 75 HHV-8 genes are collinear with their EBV homologs. Among these 54 genes, the average amino acid identity is 35%. EBV has three forms of viral latency but HHV-8 has only one that has been identified.

Neutralizing antibodies are part of the humoral defense system against viral infection. The presence of nAb has been detected by searching for the effect of inhibition by nAb against HHV-8 viral infection in transformed dermal microvascular endothelial cells 41. By quantifying the level of viral infection by indirect immunofluorescence assay (IFA), inhibition of infection was determined by comparing the level of infection in cells obtained with HHV-8 seropositive sera as compared to the level shown by incubation with seronegative sera. When the seropositive sera was diluted at 1:10 or 1:50 there was significant inhibition compared to the seronegative controls (P = 0.036). However, at a 1:500 dilution, the inhibitory effects of the sera disappeared. The nAb were found in the IgG fraction as shown by depletion of IgG antibody with protein A, which reversed the inhibitory effect.

Similarly, the presence and effect of nAb in the context of HHV-8 infection were investigated by measuring the infectivity in the 293 culture cell line 42. Kimball et al. also discovered that the nAb were found in the IgG fraction and that compliment was not required for the neutralization. Importantly, their study found that those patients with KS had significantly lower nAb titers than other groups, independent of their HIV status. This suggested a possible role for nAb in the prevention of progression from latent asymptomatic HHV-8 infection to KS disease. They state that the positive effects of nAb were independent of CD4+ counts.

In contrast to these two reports, Inoue et al. observed the effects of nAb action, but concluded that nAb do not affect the progression to KS 43. These antibodies were found in both KS+ and KS- groups with prevalences of 24% and 31%, respectively, but there was no significance in the difference (P = 0.64). This conflicting finding could perhaps be explained by the specific cohorts used. Other possibilities are the use by Inoue et al. of a colorimetric reporter system and their choice of cutoff at 30% neutralization; where as Kimball et al. used 50% inhibition as the cut off 42. Additional discussion of HHV-8 antibody responses can be found in Sections 7 and 8.

Cell mediated immunology studies of HHV-8 have indicated that there are specific cytotoxic T-lymphocyte (CTL) responses against the virus. In an investigation of five cases of HIV negative subjects that seroconverted to HHV-8, Wang et al. explored the CD8+ T-cell response to five HHV-8 lytic proteins and found that CD8+ T-cells are involved in the control of primary HHV-8 infection 44. They found that there were no major changes in the numbers of T-cell phenotypes or activation of T-cells, which differed from primary EBV infection that usually produces global increases in the numbers of T-cells. There was also no suppressive effect on other T-cell specificities as seen with EBV infection. They observed distinct CD8+, HLA class I restricted responses and increases in the interferon-gamma (IFN-γ) response to at least three of the five lytic antigens in each of the five subjects. No antigen was dominant in the elicited T-cell response. They observed that HHV-8 antibody titers to lytic IFA proteins paralleled the cytolytic responses. The CD8+ reactivity declined after several years possibly because of the lack of stimulation; the normal biology of HHV-8 is to enter a more latent state after primary infection. More T-cells produced a response of INF-γ production as opposed to CTL precursor production, but neither response was as strong as that observed when the T-cells were challenged with the HCMV pp65 antigenic protein. Osman et al. investigated HLA class I restricted CTL activity directed against the HHV-8 K8.1 lytic antigen 45. They also investigated an additional lytic protein (K1) and one latent protein (K12) as antigens. Chromium release assays showed that CTL reactivity was detected against all three proteins, but not every patient had reactivity to all three antigens. Specific HLA alleles were able to present more than one of the viral proteins; e.g., HLA B8 could present all three antigens. Most patients with KS and were HIV+ did not have CTL responses indicative of compromised cellular immune systems. In one patient, whose KS had resolved under HAART therapy, CTL activity was restored. In general, these investigators showed that higher titers against HHV-8 LANA1 (Orf 73), i.e., more severe KS, correlated with less CTL response.

In a study of seroconversions in Amsterdam, Goudsmit et al. found that CD4+ T-cell levels did not affect the rate of seroconversions, but once HHV-8 infection had occurred, a decline in CD4+ cells was associated with increasing reactivity against the Orf 65 antigen 46. Similar findings have been reported by Kimball et al. where persons with KS have higher levels of anti-HHV-8 antibodies and lower CD4+ counts than those without KS, but where both populations have HIV infection 42. This suggests that viral replication had increased in the context of a more limited CD4 response. Recent investigation 47 has shown that NK cell function is important for the control of latent HHV-8 infection and abrogation of this important immune response can lead to more progressive KS disease.

Using peripheral blood mononuclear cells (PBMCs) culled from KS patients and grown in culture, Monini et al. showed that reactivation of HHV-8 required at least the inflammatory cytokine (IC) INF-γ 48. They observed that both B-cells and monocytes latently infected with HHV-8 responded to this IC with induction of lytic replication. They proposed that increases in HHV-8 viral load are due to the reactivation of the virus after exposure to INF-γ. They also proposed that a likely scenario of KS pathogenesis is the recruitment of circulating monocytes into peripheral skin tissues, where upon exposure to ICs, their latent HHV-8 genomes enter into the lytic phase. The monocytes then rupture and free virus is available to infect local tissues. The monocytes might also differentiate into macrophages or spindle cells after exposure to the ICs and form the basis of latent HHV-8 infection in the tissues.

Reactivation is possible in the context of autologous peripheral blood stem cell transplantation. Luppi et al. 49 presented a case report that showed HHV-8 viral load in the serum of the transplant patient concomitant with fever, rash, diarrhea, and hepatitis some 17 days after the transplant. The patient had lytic antibodies before and after the transplant indicating a reactivation event.

A number of studies 4950515253545556 have investigated by molecular methods the presence of HHV-8 virions, as evidenced by the presence of viral DNA in body fluids and tissues of several at-risk populations (Table 1). PBMCs were the most commonly studied sample site, but a number of others, including serum or plasma, semen, saliva, and stool have been investigated (Table 1). PCR sensitivities were below 100 copies, although some studies used nested PCR 52 or Southern blotting 50.

At least four investigators used the K330 PCR as originally developed by Chang et al. 1. Five articles described testing KS patients 5051525455 and another five 5051525556 compared HIV+ and HIV- subjects for the presence of HHV-8. Grandadam et al. 53 investigated multicentric Castleman's disease (MCD) in HIV+ patients and Luppi et al. 49 followed the unique case of a viral reactivation. For persons with KS, significant differences were found between sample sites; the HHV-8 prevalence was higher in KS lesions over that found in peripheral blood mononuclear cells (PBMCs), which were about equal in prevalence to saliva (Table 1). These three sites were better for finding the presence of HHV-8 rather than using plasma (P <10^-6; P = 0.054; P ≤ 0.02, respectively). For HIV+ persons, saliva and PBMCs were equivalent (P = 0.539) but both had a significant greater frequency of positive samples than were found in plasma (P = 0.016 and P = 0.031, respectively). Analysis of HIV- persons showed that saliva contained significantly more viral sequences than either PBMCs or plasma (P = 0.001 and P = 0.0006, respectively), which were commensurate with each other (P = 0.476).

It is noteworthy to add that several authors have observed the detectable presence of HHV-8 DNA to be intermittent 49515758. Perhaps this has contributed to the overall lack of sensitivity of PCR in detecting HHV-8 infection. In keeping with this observation, Simpson et al. 59 stated, "...KSHV genomes were detected in peripheral blood monocyte DNA from KS patients less frequently than antibodies to either KSHV antigen in serum". Smith et al. 60 added that, "Overall, our serologic assay appeared more sensitive than PCR analysis of PBMC for the detection of HHV-8 infection". This last statement was reiterated by other authors (e.g. Angeloni et al. 61, Campbell et al. 62). HHV-8 viremia is described at more length in Section 8, HHV-8 Diagnostics.

The transmission of HHV-8 through sexual activities has been documented 74; men with homosexual behaviors showed a 38% prevalence of HHV-8 as compared to 0% of men with no such activity. The increased prevalence correlated with the presence of sexually transmitted diseases (STD) and the number of male sexual partners. The presence of both HIV and HHV-8 produced a 10-year probability of 50% for developing KS 74.

Transmission from male genital secretions, specifically semen, is unlikely due to the low prevalence of detectable HHV-8 in semen samples obtained from both HIV+ or HIV- persons 525556. In a study of women with KS from Zimbabwe, between 28% and 37% had detectable HHV-8 DNA in their vaginal or cervical samples 75, but HHV-8 DNA was not found in any of the women without KS, even those with HHV-8 seropositivity. A possible explanation why perinatal transmission is infrequent in prevalence studies might be that transmission is limited to immunocompromised mothers where titers might be higher 75.

HHV-8 DNA is found most frequently and with increased viral burdens in saliva or other oral samples 56. Sexual practices that include oral sex could therefore increase the possibility of transmission. Persons having STDs, such as syphilis and HIV, have an increased risk for greater HHV-8 prevalence 76. However, in a study of 1,295 women in four USA cities, Cannon et al. did not find an association between the number of sex partners or engagement in commercial sexual practices to be a risk for increased HHV-8 prevalence 76.

Identification of HHV-8 in blood donors 5877 has raised concern about the safety of the blood supply. Other reports 78 have tempered the concern of blood borne transmission after observing no transmission in 18 recipients of HHV-8 seropositive blood components. However, because of the small sample size, additional studies are required for this low prevalence population. In a multicenter study of 1,000 blood donors, approximately 3% of blood donors were considered seropositive, but none of the 138 total seropositive samples had detectable HHV-8 DNA in their PBMCs 79. Without detectable virus, the possibility of infectious transmission seems remote.

However, blood-borne transmission seems to occur, but rarely. Two epidemiological markers for blood borne viral infection, HCV positivity and daily-injected drug use, were associated with increased HHV-8 infection in four large groups of women in the USA 76. However, the overall prevalence of HBV and HCV among irregular drug users was higher than found with HHV-8, indicating a lower relative frequency of transmission of this herpesvirus.

Evidence that HHV-8 can be transmitted in populations of intravenous drug users (IVDU) and those HCV+, shows that transmission via blood is possible, albeit with difficulty 80. Larger studies are required to determine if HHV-8 is a true threat to the blood supply. Such studies will be difficult to conduct due to the difficulty in detecting infectious virus in healthy individuals, the lack of culture methods to tests for cytopathic effect, and the anonymous nature of blood donations, which does not allow for follow up testing.

Important risk factors for transmission of the virus are a spouse's seropositivity and maternal seropositivity 81. Although spousal seropositivity could include sexual transmission, transmission to children precludes this route, indicating more casual transmission is possible. Horizontal asexual transmission within families has been observed by other investigators 82. Vertical transmission from mother to child at or before birth is also infrequent with few children from HHV-8 infected mothers showing HHV-8 sequences in their PBMCs at birth 8384. In a study of the presence of HHV-8 DNA in matched pairs of breast milk and saliva from the same mother, no HHV-8 sequences were found in the breast milk, but 29% of the saliva samples had HHV-8 DNA; therefore nursing of infants appears unlikely to be a route of infection 85, although, another study seemed to contradict this finding 86.

Of all anatomic sites, HHV-8 DNA is found most frequently in saliva, which also has higher viral concentrations than other secretions 56. For this reason, it has been hypothesized that saliva could be the route of casual transfer of infectious virus among family members. It has been hypothesized that customarily licking an insect bite, such as from a mosquito, could transfer the virus 87.

Identification of HHV-8 primary infection has been difficult due to the low incidence of infection in most populations studied, and because of the lack of known defining features. By using a diagnosis of exclusion and the temporal occurrence of symptoms and diagnostic criteria, limited studies have suggested several defining clinical sequelae of HHV-8 primary infection. In 15-year longitudinal study of >100 HIV negative men to study the natural history of primary HHV-8 infection, five cases of HHV-8 seroconversion were identified 44. The effects of HHV-8 primary infection were explored in the absence of HIV coinfection and no debilitating disease was observed in the five seroconverters. Four patients exhibited clinical symptoms, which ranged from mild lymphadenopathy and diarrhea to fatigue and localized rash. These symptoms were significantly associated with HHV-8 seroconversion when compared to the 102 seronegative subjects who remained well.

Organ transplantation is another clinical setting for primary infection. In a patient receiving a renal transplant, bone marrow failure was associated with a syndrome of fever, marrow aplasia, and plasmacytosis 104. The patient did not present with KS, but HHV-8 sequences were detected by PCR after transplantation and at the presentation of symptoms; the patient did not survive. This limited experience suggests that in the context of immunosuppression, primary infection can be lethal, but in healthy individuals, the infection presents with flu-like symptoms.

KS was first described by Moritz Kaposi in the 1870s 105 and was described as an aggressive tumor affecting patients younger than those currently observed. For all epidemiological forms of KS, the tumor presents as highly vascularized neoplasm that can be polyclonal, oligoclonal, or monoclonal. It's antigenic profile suggests either endothelial, lympho-endothelial, or macrophage origins 106. Although the four epidemiological forms of KS have different clinical parameters, such as anatomic involvement and aggressiveness of the clinical course, they have HHV-8 infection in common with indistinguishable histopathology 107. It is therefore believed that this transforming virus is the causative agent of KS and that HHV-8 fulfills Hill's criteria for causing KS 108109.

HIV infection substantially increases the risk for development of KS, and therefore, the incidence of KS has increased substantially during the HIV pandemic, particularly in younger HIV-infected patients 110. Striking differences in risk for acquiring AIDS-KS exist between different HIV transmission groups, varying from a high of 21% for homosexual men to a low of 1% for men with hemophilia. Women who acquired HIV infection by heterosexual contact with bisexual men were also at an increased risk for developing AIDS-KS 110. Although the incidence of KS has decreased recently with the advent of highly active anti-retroviral (HAART) therapy, the appearance of drug resistant strains of HIV raises concern for a re-emergence of KS cases.

Browning et al., using a cell culture detection method, observed that the characteristic spindle cells of KS are present in the peripheral blood of patients presenting with KS; more importantly, these cells were found in the blood of HIV+ homosexual men, who are at higher risk for developing KS, than HIV+ IVDUs 102.

The first strong evidence that human herpes virus 8 (HHV-8) was the etiological agent of KS came from the use of a novel molecular technique, representational difference analysis (RDA) 1. This complex molecular method identified viral molecular sequences in KS tumor tissue that were not present in paired normal tissue from the same individual 1. The presence of nucleic acid sequences of the virus in tissues from all forms of KS 111 throughout the world, and the demonstration of antibodies to HHV-8 in KS patients from a number of serologic studies 112 has supported the association of this virus with KS. Because of its prominent association with KS, the virus is often referred to as Kaposi's sarcoma-associated herpesvirus or KSHV.

Proof of HHV-8's etiology in KS comes from the detection of HHV-8 nucleic acids in KS tissues but not in healthy tissues, from sero-epidemiological and molecular studies showing correlations to the risk of developing KS and progression of KS disease. The detection of antibodies to lytic HHV-8 antigens can be used as a predictor of development of KS 113. Prospective studies of persons who subsequently developed KS, documented the appearance of infection more than 24 months prior to tumor development 114. Data have shown that infection of primary endothelial cells with HHV-8 causes long term proliferation and transformation 115. HHV-8 is detectable in the spindle cells of all forms of KS and in the nearby in situ endothelial cells 27.

First identified as a subset of body-cavity-based lymphomas (BCBL), PELs contain HHV-8 DNA sequences 23. These lymphomas are distinct from malignancies that cause other body cavity effusions. PELs are characterized by several pathological features: 1) They do not exhibit Burkitt lymphoma-like morphology and do not have c-myc gene rearrangements; 2) They have a distinctive morphology comparable to large-cell immunoblastic lymphoma and anaplastic large-cell lymphoma; 3) They occur frequently in men; 4) They present initially as a lymphomatous effusion and remain localized to the body cavity of origin; 5) They express CD45 with frequent absence of B-cell associated antigens; 6) They exhibit clonal immunoglobulin gene rearrangements; 7) They can contain Epstein-Barr virus; 8) They lack oncogene rearrangements in genes such as bcl-2 and p53. Finally, patients with PELs, especially in the context of AIDS, invariably are infected with HHV-8 23131. PEL cell lines have 50–150 copies of HHV-8 episomes per cell 8132133134135136.

Divining the association of PELs with HHV-8's etiology has been difficult, because most PELs occur in the context of HIV infection, and the PELs account for only 0.13% of all AIDS malignancies in AIDS patients in the USA 137. Importantly, PELs occur with an increased frequency in patients with prior KS 125. In non-AIDS patients, the disease has been termed "classic" PEL by Ascoli et al. 138 where it presents in HIV negative patients, but with similar risk factors as CKS.

HHV-8 has been found variably in association with MCD. MCD is a rare polyclonal B-cell angiolymphoproliferative disorder for which vascular proliferation has been found in germinal centers. It presents in heterogeneous forms both clinically and morphologically 139. However, most of the B-cells in the tumor are not infected with HHV-8, and the HHV-8 infected cells are primarily located in the mantel zone of the follicle 140. It is thought that uninfected cells are recruited into the tumor through HHV-8 paracrine mechanisms, such as vIL-6 66, a known growth factor for the tumor. More than 90% of AIDS patients with MCD are HHV-8 positive, whereas MCD in the context of no HIV infection has a HHV-8 prevalence of approximately 40% 141. Because of it rarity, MCD is difficult to closely associate statistically with HHV-8.

No single treatment has been found to be completely efficacious for HHV-8 infection. Anti-herpetic drugs such as foscarnet, ganciclovir, cidofovir, and acyclovir inhibit the viral DNA polymerase 107 which, therefore, only allows treatment for replicating viruses in the lytic phase of infection; latent viruses are unaffected. For example, although cidofovir was effective in vitro against BCBL-1 cells 160, intralesional injections were not helpful in reducing the KS tumor burden 161.

Chemotherapy and/radiotherapy are successful treatments for CKS but HHV-8 DNA has been shown to remain at the site of the healed lesion 162. This might explain the observed reoccurrences of CKS. Treatment for AIDS-KS has centered on HAART. Studies have shown marked decreases in the incidence of AIDS-KS since the use of HAART 163. However, this reduced risk has been only with triple therapy, and not double or single anti-HIV drug therapy 163. Additionally, HAART seems to have the best effect on early stage AIDS-KS 164165; nonetheless, an 81% reduction in death due to AIDS-KS was observed though HAART 164.

Finally, because HHV-8 can be transferred from organ donor to recipient, the possibility exists that CTLs derived from the donor can be harnessed to provide immunotherapy for the recipient 100. This has been shown to be an effective treatment for PTLD in the context of EBV reactivation after bone marrow donation 166.

The serologic prevalence of HHV-8 infection has been explored in most continents worldwide and in different populations at different levels of risk of HHV-8 infection. It should be noted that the comparisons of prevalence are limited by whether antibodies to latent or lytic HHV-8 antigens were detected and the test formats used.

From DNA sequence analysis of distinct loci derived from 60 HHV-8 isolates, the clustering of four major HHV-8 viral subtypes was discovered 206. These subtypes, A, B, C, and D are based upon DNA sequence derived from the K1 gene, a glycoprotein with transforming properties 207208, and they exhibit 30% amino acid (aa) variability. These aa substitutions result from an 85% nucleotide substitution rate in this highly variable gene. The four subtypes were further divided into another 13 clades by Hayward 206. The A1, A4, and C3 variants were predominant in the US AIDS KS samples, but the B variant was predominant in samples from Africa. C variants were observed from samples from Saudi Arabia and Scandinavia. The D subtype was uncommon and was found only in classic KS patients in the Pacific region. Another gene, K15, showed two different alleles (P and M), but these allelic types were not associated with the K1 subtypes 206. These different genotypes have been investigated to explain the possible pathogenic and epidemiologic variation seen with HHV-8 infection 125. Studies that are more recent have expanded upon previous work and have shown that the K1 locus can be divided into six subtypes with 24 clades showing strong linkage to the geographic origin of the particular isolate. Data have shown that subtypes A and C are prevalent in Europe, the U.S.A., and northern Asia. Subtypes B and A5 predominate in Africa and the D variant is found in the Pacific. Subtype E has been discovered in Brazilian Amerindians and a unique subtype Z was found in Zambia 125. In a recent study, Whitby et al. characterized the K1 hypervariability from general populations in South America and Africa: i.e., those without any obvious symptoms of HHV-8 infection 179. Amerindians from Ecuador carried the E subtype, in keeping with previous studies from South America. In Botswana, subtypes B and A5 were exhibited by subjects from the Bantu and San tribes, similar to the subtypes found there from KS patients. These results show that the same HHV-8 viral strains from similar geographic regions can be found in both diseased and non-diseased individuals, suggesting that there is no association between certain genotypes and disease.

Immunofluorescent observations that PEL cells exhibited a distinct nuclear immunofluorescence after challenge with antisera from KS patients, led to the identification of Orf 73 as the gene responsible for the latency associated nuclear antigen-1 (LANA1) 209210211. Early gene alignments had suggested that Orf 73 was an immediate early gene with 51% similarity to the Orf 73 of HVS 19. Studies have since shown that LANA1 is a 222–234 kDa protein that is expressed in the majority of nuclei in KS spindle cells 211212; however, the LANA1 protein expression is variable 211 and can depend upon the clinical stage of the KS tumor 213. The immunodominant epitope has been mapped to the C-terminal domain of the protein 210. The gene is under latent control as evidenced by reduction in Orf 73 mRNA after chemical induction of the viral lytic phase 210. The antigenicity of the recombinant LANA1 protein has been shown by Western blot; over 70% of HHV-8 IFA seropositive sera were LANA1 positive in the Western blot 210211.

Orf 65 was identified by Russo et al. 19 as a lytic capsid protein with less than 60% similarity to similar capsid proteins from HVS and EBV, but is not cross-reactive with HVS and EBV capsid proteins 59214215216. Orf 65 has been shown to be the smallest component of the HHV-8 capsid with a predicted basic isoelectric point of 9.6, similar to other herpesviruses 217. Because of its embossed structural position on the capsid, Orf 65 might be involved in interactions with the viral tegument and cellular proteins upon infection 218. First cloned in bacteria by Simpson and colleagues 59 and subsequently by others 215, Orf 65 is a highly antigenic 18–22 kDa protein against which more than 81% of KS patients are seroreactive 59215. The dominant eight amino acid epitope has been mapped to the C-terminus, and allowed development of a peptide assay with reactivity in 90% of the KS samples tested 216.

Originally identified as a single gene locus 19, research has since shown that K8.1 is derived from spliced transcripts 219 for which the transmembrane sequence is appended 220. This glycoprotein is unique to HHV-8 and is a TPA-inducible lytic protein 221. On Western blots from induced PEL cells, it measures between 35–40 kDa with the characteristic smear of a glycoprotein 221. Immunoelectron microscopy suggests that the virion acquires the K8.1 glycoprotein at the cell plasma membrane while budding from the host cell 222. Two transcripts are produced, K8.1A and K8.1B, of which K8.1B is the shorter by dint of an internal deletion of 61 amino acids. K8.1A, casually referred to as K8.1, is very antigenic, with 97% of HIV+, KS+ patients having antibodies directed against it on Western blot; in HIV+, KS-, persons 61% showed reactivity 219.

Orf 25 and Orf 26 code for other major and minor HHV-8 capsid proteins, respectively, and were investigated for their diagnostic utility 223. Orf 25 possesses 68% identity to the EBV BCLF1 major capsid protein and exhibited considerable cross-reactivity to EBV+ sera and was not used further in their studies. However, Orf 26 has only 49% identity to its EBV gene homologue and showed no cross-reactivity 223. Only one third of KS patients were reactive to Orf 26, although some exhibited an increase in IgM and IgG reactivity 15 months prior to KS disease.

The Orf 59 protein is another HHV-8 protein that has shown modest diagnostic importance in a few investigations. This gene has about 50% similarity with its HVS and EBV homologues and is presumed to be a DNA replication protein in those viral systems 19. Orf 59 is a 50 kDa protein with characteristic early-late lytic expression patterns seen for other viral proteins necessary for viral DNA replication 224. The protein has been localized to the nuclear membrane via IFA and is observed in approximately 30% of induced PEL cells, but in less than 8% of uninduced cells 224. The Orf 59 gene product, processivity factor-8, has been shown to be present in AIDS-KS tumors (50%) although perhaps not in as many spindle cells as Orf 73 225. Approximately 30% of AIDS-KS patients had antibodies against this antigen 226. Orf 59 might be helpful in identifying aggressive KS disease 225226.

Presently, the diagnosis of KS requires clinical and histologic evaluation; however, the increasing documentation of its association with HHV-8 has raised the important possibility of being able to predict disease occurrence by demonstrating HHV-8 infection 55. Additionally, there is a need to develop sensitive and specific serological assays to detect antibodies to HHV-8 for possible blood bank screening, assisting in clinical diagnosis, and in research to facilitate the understanding of the scope of this virus's association with rare, but nonetheless life threatening malignancies. HHV-8 infection can be identified by polymerase chain reaction in tissues and in cells; however, amplification methods are expensive, time consuming, and have been shown to be lacking in sensitivity for easily accessible diagnostic specimens such as plasma and PBMCs 177. Alternatively, the testing for specific antibodies to HHV-8 offers a simple, inexpensive, and effective means to document infection and a help to define the relationship between infection and disease progression and yield insight into pathogenic mechanisms.

Currently, four methods have been used to demonstrate antibodies to HHV-8: enzyme-linked immunosorbant assay (ELISA), immunofluorescent assay (IFA), Western blot, and immunohistochemistry (IHC). Detection of infection and determination of seroprevalence can be dependent upon which test is selected 227. ELISA methods vary according to the HHV-8 antigens used and whether they are recombinant antigens, viral lysates, or synthetic peptides. IFA methods incorporate virally-infected cell lines, either latently infected with expression of LANA1, or cells that express lytic antigens following chemical induction (i.e., those representing viral replication). The Western blot technique utilizes electrophoretically separated virally infected cell lysates or whole viral lysates, with transfer to nitrocellulose and then subsequent detection of reactive antigens; it has the advantage of identifying the presence of antibodies to specific antigens. IHC on fixed cells and tissue allows to determination of which cells harbor the virus in vivo and semi-quantitative analysis of infected sell type to help learn more about pathogenesis. IHC is also useful to confirm or rule out the clinical diagnosis of KS. These tests are explored in the following sections.