A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults.

Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

A replication-competent adenovirus assay for E1-deleted Ad35 vectors produced in PER.C6 cells.

The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.

Human adenovirus type 35 vector for gene therapy of brain cancer: improved transduction and bypass of pre-existing anti-vector immunity in cancer patients.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Improved gene delivery to intestinal mucosa by adenoviral vectors bearing subgroup B and d fibers.

A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

[The molecular epidemiology of HIV-1 in Belarus in (1996-2004): a predominance of subtype A variants and circulation of subtype B variants].

To study the molecular epidemiology of HIV-1 in Belarus, the genetic sequences of HIV-1 variants were obtained from 50 infected persons, which represented the main stages, risk groups, and geographic areas of the epidemic. The env and gag sequences were studied for HIV-1 variants from 31 persons, the env sequences were for HIV-1 variants from 18 persons, and the gag sequence was for HIV-1 variant from 1 person. Phylogenetic analysis indicated that the sequences of HIV-1 variants from 46 persons were homogenic and evolutionally closely related to IDU-A strains specific for other epidemics in the former Soviet Union are dominating in the epidemic in Belarus. Circulation of epidemiologically unrelated subtype B viruses was also established.

An automatic hinge system for leg orthoses.

This paper describes a new automatic hinge system for leg orthoses, which provides knee stability in stance, and allows knee-flexion during swing. Indications for the hinge system are a paresis or paralysis of the quadriceps muscles. Instrumented gait analysis was performed in three patients, fitted with this new hinge system in a knee orthosis. The orthosis proves to satisfy the patients allowing them to walk without a knee-lock system.

Adenovirus fibre exchange alters cell tropism in vitro but not transgene-specific T CD8+ immune responses in vivo.

Gene transfer with recombinant adenoviruses (rAds) is a powerful means of inducing an immune response against a transgene product. However, little is known about the mechanisms that underlie the induction of the immune response after intramuscular inoculation of adenovirus and, in particular, the relative role of the different cell types transduced. Several studies have suggested that CD8+ cytotoxic T lymphocyte responses elicited after inoculation of adenoviruses (Ads) are induced both by direct transduction of antigen presenting cells (APCs) and by cross-priming. In the present study, a library of fibre-chimeric rAds was screened in order to identify rAds with distinct capacities to express transgene product in murine cell types naturally found in muscle, i.e. myoblasts, endothelial cells (both representing non-APCs) and dendritic cells (representing APCs). Four selected pseudotypes, differing in their ability to infect muscular cells were used to immunize C57BL/6 mice. The relationship between the capacity to transduce non-APC or APC in vitro and the ability to induce humoral and cellular responses against the beta-galactosidase antigen after intramuscular inoculation were studied. Results indicate that CD8+ T cell responses against the beta-galactosidase antigen were similar after inoculation of the four viruses, thus revealing no direct relationship with their ability to transduce myoblasts, endothelial cells or dendritic cells in vitro.

A rapid method for immunotitration of influenza viruses using flow cytometry.

Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.

Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes.

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.

In vitro assay for site-specific proteases using bead-attached GFP substrate.

Site-specific proteases, which catalyze cleavage of peptide bonds in specific amino acid sequences of target proteins, play important roles in various biological events of many living organisms. In humans, disruption in regulation of these site-specific proteases can lead to pathological consequences. Here, we report a simple in vitro assay for enzymatic activities of site-specific proteases. This assay system employs a protein substrate molecule that is comprised of (i) His-tag binding module, (ii) cleavage sites, and (iii) green fluorescent protein (GFP) detection module. In this study, prostate-specific antigen (PSA) and Thrombin-specific cleavage sites were introduced into the substrate molecules. The overexpressed GFP substrate protein was purified with the aid of Ni++-charged magnetic beads. On cleavage by either PSA or Thrombin, GFP was released from the bound magnetic beads, enabling a direct measurement of the cleaved product by fluorescence. Detection sensitivity, as well as the kinetics of reaction of PSA cleavage with the GFP substrate, was similar or better than commercially available PSA fluorogenic peptide substrate. This bead-attached GFP substrate was also used for an inhibition assay using a competitive inhibitor of Thrombin. In conclusion, this assay offers a simple fluorescent method for monitoring the activity of the site-specific proteases. Furthermore, this system provides flexible means of incorporating varying sizes of flanking sequences adjacent to the cleavage site, which can be essential for studying the regulatory macromolecular interactions between proteases and their substrates.

Kaposi's sarcoma and human herpesvirus 8 infection do not protect HIV-1 infected homosexual men from AIDS dementia complex.

OBJECTIVE: To examine the association between Kaposi's sarcoma (KS), human herpes virus 8 (HHV8) and AIDS dementia complex (ADC). DESIGN: A total of 599 HIV-1 infected homosexual men participated in a prospective cohort study (Amsterdam, 1984-1996). METHODS: The risk for ADC in patients with prior KS or HHV8 infection was estimated using the Cox proportional hazards method with adjustments for antiretroviral medication and low CD4 cell counts. RESULTS: Of the 599 participants, 290 (48.4%) had HHV8 antibodies, 99 (16.5%) had KS and 30 (5.0%) had ADC. ADC was diagnosed in 5.2% of participants with KS and 5.0% of those without KS, and in 4.8% of HHV8 seropositive compared to 5.2% seronegative individuals and thus was not associated with KS or HHV8 infection. Using a time-dependent Cox proportional hazards analysis with the date of KS as risk factor, the risk for ADC was 2.7 [95% confidence interval (CI), 0.92-7.96; P = 0.07) and when only definite ADC was considered it was 3.5 (95% CI, 1.00-12.26;P = 0.05). After adjusting for decreases in CD4 cell count and use of medication, the hazards ratio for participants with KS to develop ADC was 2.0 (95% CI, 0.66-5.77; P = 0.23) and 2.6 (95% CI, 0.73-9.12; P = 0.14), respectively. HHV8 seropositivity, adjusted for the same variables, showed a risk for ADC of 0.85 (95% CI, 0.41-1.77;P = 0.66) and for definite ADC 0.69 (95% CI, 0.27-1.73; P = 0.42). The expected neuroprotective effects of antiretroviral medication were observed. CONCLUSIONS: KS or HHV8 does not significantly influence the risk for developing ADC in a group with a uniform risk for developing KS therefore we recommend caution in searching for a KS-associated or HHV8-derived therapy for ADC.

Evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses.

The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary groups of parvoviruses from vertebrates: (i) the human helper-dependent adeno-associated virus (AAV) serotypes 1 to 6 and the autonomous avian parvoviruses; (ii) the bovine, chipmunk, and autonomous primate parvoviruses, including human viruses B19 and V9; and (iii) the parvoviruses from rodents (except for chipmunks), carnivores, and pigs. Each of these three evolutionary groups could be further subdivided, reflecting both virus-host coevolution and multiple cross-species transmissions in the evolutionary history of parvoviruses. No parvoviruses from invertebrates clustered with vertebrate parvoviruses. Our analysis provided evidence for negative selection among parvoviruses, the independent evolution of their genes, and recombination among parvoviruses from rodents. The topology of the phylogenetic tree of autonomous human and simian parvoviruses matched exactly the topology of the primate family tree, as based on the analysis of primate mitochondrial DNA. Viruses belonging to the AAV group were not evolutionarily linked to other primate parvoviruses but were linked to the parvoviruses of birds. The two lineages of human parvoviruses may have resulted from independent ancient zoonotic infections. Our results provide an argument for reclassification of Parvovirinae based on evolutionary relationships among viruses.

Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984-1997): analysis of seroconversions to ORF65 and ORF73.

We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections.

Primate genus Miopithecus: evidence for the existence of species and subspecies of dwarf guenons based on cellular and endogenous viral sequences.

Sequence data from the mitochondrial 12S rRNA gene were combined with endogenous retrovirus sequences to study the position of the genus Miopithecus in the primate tree. The mitochondrial sequences indicated that Miopithecus is a true genus distinct from Cercopithecus, although talapoin monkeys are commonly referred to as dwarf guenons. The existence of two species of dwarf guenons, suggested by differences in coat color, pigmentation, and geographic location, was supported by substantial mitochondrial 12S rRNA gene divergence. In line with the informal proposal of J. Kingdon (1997, "The Kingdon Field Guide to African Mammals," Academic Press, London), we use the names Miopithecus talapoin for the southern, darker species and Miopithecus ougouensis for the northern, lighter-colored monkeys. Different 12S rRNA gene haplotypes found in M. ougouensis individuals suggest the possible existence of additional subspecies. Simian endogenous retrovirus (SERV) strain 23. 1 proviruses were introduced in the primate germ-line after the Cercopithecinae split from the Colobinae, estimated at around 9-14 million years ago. SERV sequences were used for timing of divergence events in Cercopithecinae and confirmed the close relationship between the genera Cercopithecus and Miopithecus, which was only weakly supported by the more variable mtDNA sequences in a distance analysis, demonstrating the utility of these pseudogenes in phylogenetic grouping.

Risk factors for human herpesvirus 8 seropositivity and seroconversion in a cohort of homosexual men.

Sexual and nonsexual modes of transmission of human herpesvirus 8 (HHV8) have been suggested, but specific routes remain unclear. Therefore, the objective of this study was to assess risk factors for HHV8 seropositivity and determine specific sexual practices associated with HHV8 seroconversion. Sera from 1,458 homosexual men (Amsterdam Cohort Study, 1984-1996) were tested for antibodies to HHV8 with a modified version of an enzyme immunoassay, using recombinant HHV8 lytic phase capsid (ORF65) and latent phase nuclear (ORF73) proteins. HHV8 seroprevalence at study entry was 20.9% (305/1,458); was highest among those with positive human immunodeficiency virus (HIV) status, no steady partner, and southern European or Latin American nationality; and increased with older age and higher number of sexual partners. During follow-up, 215 men seroconverted for HHV8 (incidence: 3.6/100 person-years). Both prevalence and incidence rates remained more or less stable during the study period. Orogenital insertive sex (odds ratio (OR) = 5.95; 95% confidence interval (CI): 2.88, 12.29) or orogenital receptive sex (OR = 4.29; 95% CI: 2.11, 8.71) with more than five partners in the past 6 months, older age (OR = 2.89; 95% CI: 1.13, 7.34, when older than 45 years), and preceding HIV infection (OR = 2.47; 95% CI: 1.53, 3.99) were independent predictors for HHV8 seroconversion. The authors found strong evidence for orogenital transmission of HHV8 among homosexual men.

Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2 herpesvirus lineage among old world primates?

Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens.