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Risk for human exposure to bovine spongiform encephalopathy (BSE)-inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received >5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7-5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7-10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown.

The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized.We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro.The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC503μg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV.N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.

BACKGROUND: The cellular prion protein (PrPc) plays a central role in prion diseases such as variant Creutzfeldt-Jakob disease. This disease can be transmitted by blood transfusion. However, the exact kinetics of blood infectivity and the blood fraction carrying infectivity have not yet been identified. STUDY DESIGN AND METHODS: Simian PrPc epitopes were mapped by flow cytometry using monoclonal antibodies (MoAbs). A whole blood/no wash protocol was established, validated, and applied to investigate peripheral blood cell–associated PrPc expression profiles in bovine spongiform encephalopathy (BSE)-infected cynomolgus monkeys and age-/sex-matched controls. In addition, physiologic expression patterns on blood cells and in lymphoid tissues were determined. RESULTS: In BSE-infected macaques, blood lymphocyte–associated PrPc fluorescence gradually increased years before the onset of clinical signs (pF test

In this study we investigated the efficacy of a multigenic DNA prime/modified vaccinia Ankara (MVA)boost vaccine approach, followed by mucosal challenge with highly pathogenic simian-human immunodeficiency virus (SHIV) 89.6P, using different routes for vaccine delivery. After three times of DNA priming (SIVmac239, GagPol, and SHIV 89.6P Env) one vaccine group of monkeys was immunized with MVA systemically via intramuscular (IM) and intradermal (ID) application, and in another vaccine group the MVA booster immunization comprised the IM, ID, and atraumatic oral route. Although all vaccinees became infected after intra-rectal challenge with SHIV 89.6P, substantial protection as indicated by lower peak and set point viral loads and unambiguous preservation of CD4 T cells could be achieved. As we could only transiently detect low levels of neutralizing antibodies in some vaccinees, these antibodies did not seem to add to the protection in the vaccinees. Our results indicate that both preventive multigenic DNA prime/MVA booster immunization strategies promote the control of virus replication and protect from disease progression. We also demonstrated that combining mucosal and systemic vaccination mediated better protective effects compared to systemic vaccination alone.

In both human immunodeficiency virus-infected humans and simian immunodeficiency virus (SIV)-infected macaques, genes encoded in the major histocompatibility complex (MHC) class I region are important determinants of disease progression. However, compared to the human human lymphocyte antigen complex, the macaque MHC region encodes many more class I genes. Macaques with the same immunodominant class I genes express additional Mhc genes with the potential to influence the disease course. We therefore assessed the association between of the Mhc class I haplotypes, rather than single gene variants, and survival time in SIV-infected rhesus macaques (Macaca mulatta). DNA sequence analysis and Mhc genotyping of 245 pedigreed monkeys identified 17 Mhc class I haplotypes that constitute 10 major genotypes. Among 81 vaccination-naive, SIV-infected macaques, 71 monkeys carried at least one Mhc class I haplotype encoding only MHC antigens that were incapable of inducing an effective anti-SIV cytotoxic T lymphocytes response. Study of these macaques enabled us to relate individual Mhc class I haplotypes to slow, medium and rapid disease progression. In a post hoc analysis, classification according to disease progression was found to explain at least 48% of the observed variation of survival time.

Serum samples were collected at the Ndola Central Hospital Cooperbelt Province Zambia from 10 adults with clinical diagnosis of AIDS. Also included were 98 successive patients attending the Sexually Transmitted Diseases (STD) Clinic none with symptoms suggestive of AIDS or ARC. The sera were tested for the presence of antibodies against HIV with 6 ELISAs: 1) H9/HTLV-III (German Primate Center); 2) ELAVIA (Pasteur); 3) Enzygnostic (Behring); 4) Wellcozyme (Wellcome); 5) env-80; and 6) anti-HIV (EIA) Roche. Sera giving positive results with any ELISA were tested further by Western blot (WB) with env-80 antigen and by radio-immunoprecipitation (RIP) with antigen from H9/HTLV-III cells. Results from RIP were the final criteria of the positivity or negativity. 20 of 108 sera were anti-HIV positive confirmed by WB and RIP. 8 of 10 patients suspected of AIDS and 12 of 98 successive unselected patients attending the STD clinic had serologically confirmed infection with HIV-1. All 20 true positive sera were detected correctly by the H9/HTLV-III ELISA but in addition 33 sera gave reactions not confirmed by WB or RIP and were classified as false positives. The ELAVIA gave 4 and Enzygnostic gave 1 false positive result but there were no false positives with Wellcozyme env-80 and anti-HIV (EIA) Roche. All 5 ELISAs other than the H9/HTLV-III failed to detect antibody in the serum of 1 STD patient in whom there was a strong reaction with gp120 alone with RIP; in addition the anti-HIV (EIA) Roche gave 1 other false negative result. The Wellcozyme and env-80 ELISAs gave identical results and both had 95% sensitivity and 100% specificity. In this small series the anti-HIV (EIA) Roche had a slightly lower sensitivity. Enzygnostic and ELAVIA had 95% sensitivity but there were false positives and lower specificity (98 and 95% respectively). The H9/HTLV- III ELISA had 100% sensitivity but only 64% specificity and 37% positive predictive value.

New features in the architecture of mammalian type C viruses, in particular knoblike surface projections and hexagonally arranged subunits on the core shell could be demonstrated by electron microscopy, taking advantage of newly developed preparation techniques. As examples, murine leukemia viruses (MuLVs) and newly isolated porcine and bovine C viruses are presented. The major proteins of a MuLV were isolated and partially characterized in chemical terms and with respect to their serological and other biological activities, such as interfering and hemagglutinating (HA) capacity. Most of the characterized proteins could be localized in particular substructures of the virion either by selective removal or isolation of electron microscopically identifiable constituents. The information obtained allowed the design of a more detailed model of mammalian C viruses. Special attention was devoted to the further characterization of interspecies antigens of mammalian C viruses. Different antigenic determinants were revealed. Their distribution allows further subgrouping of mammalian C viruses.

We have analyzed the distribution and patterns of the genetic diversity of eight Alu loci (ACE, ApoA1, PV92, TPA25, NBC27, NBC102, NBC148, and NBC182) in 1,049 individuals representing 16 populations of the Volga-Ural region (Bashkirs, Tatars, Komis, Maris, Mordvins, and Udmurts), Central Asia (Kazakhs, Uzbeks, and Uighurs), the North Caucasus (Karachays, Kumyks, Kuban Nogays, and Karanogays), and Central South Siberia (Yakuts, Kalmyks and Evenks). Geographic divide between Europe and Asia, e.g. the Ural Mountains and the Caspian Sea, can also be considered as a genetic boundary. The data indicates that the populations of the two boundary regions between Europe and Asia, the Volga-Ural region of Russia, and populations of the North Caucasus are more similar to European than to Asian populations. Finally, Siberian and Central Asian populations are genetically closely related to each other.

Diffuse large B-cell lymphoma (DLBCL) is more prevalent and more often fatal in HIV-infected patients and SIV-infected monkeys compared to immune-competent individuals. Molecular, biological, and immunological data indicate that virus-associated lymphomagenesis is similar in both infected hosts. To find genes specifically overexpressed in HIV/SIV-associated and non-HIV/SIV-associated DLBCL we compared gene expression profiles of HIV/SIV-related and non-HIV-related lymphomas using subtractive hybridization and Northern blot analysis. Our experimental approach allowed us to detect two genes (a-myb and pub) upregulated solely in HIV/SIV-associated DLBCLs potentially involved in virus-specific lymphomagenesis in human and monkey. Downregulation of the pub gene was observed in all non-HIV-associated lymphomas investigated. In addition, we have found genes upregulated in both non-HIV- and HIV-associated lymphomas. Among those were genes both with known (set, ND4, SMG-1) and unknown functions. In summary, we have demonstrated that simultaneous transcriptional upregulation of at least two genes (a-myb and pub) was specific for AIDS-associated lymphomas.

To estimate the effect of the variability of prion disease onset on primary bovine spongiform encephalopathy transmission to humans, we studied 6 cynomolgus macaques. The preclinical incubation period was significantly prolonged in 2 animals, implying that onset of variant Creutzfeldt-Jacob disease in humans could be more diverse than previously expected.

Using 40 known human-specific LTR sequences, we have derived a consensus sequence for an evolutionary young HERV-K (HML-2) LTR family, which was named the HS family. In the human genome the HS family is represented by approximately 150-160 LTR sequences, 90% of them being human-specific (hs). The family can be subdivided into two subfamilies differing in five linked nucleotide substitutions: HS-a and HS-b of 5.8 and 10.3 Myr evolutionary ages, respectively. The HS-b subfamily members were transpositionally active both before the divergence of the human and chimpanzee ancestor lineages and after it in both lineages. The HS-a subfamily comprises only hs LTRs. These and other data strongly suggest that at least three "master genes" of HERV-K (HML-2) LTRs were active in the human ancestor lineage after the human-chimpanzee divergence. We also found hs HERV-K (HML-2) LTRs integrations in introns of 12 human genes and identified 13 new hs HERV-K (HML-2) LTRs.

In HIV-infected humans and SIV-infected rhesus macaques, host genes influence viral containment and hence the duration of the disease-free latency period. Our knowledge of the rhesus monkey immunogenetics, however, is limited. In this study, we describe partial cDNA sequences of five newly discovered rhesus macaque (Mamu) class I alleles and PCR-based typing techniques for the novel and previously published Mhc class I alleles. Using 15 primer pairs for PCR-based typing and DNA sequence analysis, we identified at least 21 Mhc class I alleles in a cohort of 91 SIV-infected macaques. The results confirm the presence of multiple class I genes in rhesus macaques. Of these alleles, Mamu-A*01 was significantly associated with lower set-point viral load and prolonged survival time. Mamu-A*1303 was associated with longer survival and a “novel” Mhc class I allele with lower set-point viral load. The alleles are frequent in rhesus macaques of Indian origin (12–22%). In addition, survival probability of individual SIV-infected rhesus monkeys increased with their number of alleles considered to be associated with longer survival. The results contribute to improve the interpretation and quality of preclinical studies in rhesus monkeys.

We have developed a method of targeted genomic difference analysis (TGDA) for genomewide detection of interspersed repeat integration site differences between closely related genomes. The method includes a whole-genome amplification of the flanks adjacent to target interspersed repetitive elements in both genomic DNAs under comparison, and subtractive hybridization (SH) of the selected amplicons. The potential of TGDA was demonstrated by the detection of differences in the integration sites of human endogenous retroviruses K (HERV-K) and related solitary long terminal repeats (LTRs) between the human and chimpanzee genomes. Of 55 randomly sequenced clones from a library enriched with human-specific integration (HSI) sites, 33 (60%) represented HSIs. All the human-specific (Hs) LTRs belong to two related evolutionarily young groups, suggesting simultaneous activity of two master genes in the hominid lineage. No deletion/insertion polymorphism was detected for the LTR HSIs for 25 unrelated caucasoid individuals. We also discuss the possible research applications for TGDA research.

The value of urinary neopterin as a predictive marker for disease progression in SIV- and HIV-2-infected rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) was assessed by comparing pre- with postinfection data. Before infection stable baselines for neopterin were observed in both species with significantly higher concentrations in cynomolgus macaques than in rhesus macaques. After infection of cynomolgus macaques with human immunodeficiency virus type 2 (HIV-2) neopterin concentrations exceeded 1.2 - 4.2 times preinfection values, whereas rhesus monkeys infected with simian immunodeficiency virus (SIV) yielded concentrations of more than 8 times above baseline. Increased neopterin concentrations always preceded seroconversion. No rise of neopterin was observed in cynomolgus macaques remaining seronegative after inoculation with HIV-2, whereas after inoculation with SIV neopterin was slightly elevated in rhesus monkeys despite remaining seronegative. In animals with high virus replication a pronounced increase of neopterin levels was followed by signs of immunodeficiency. Therefore like in HIV-1-infected man, in macaques infected with SIV or HIV-2 the urinary neopterin concentration is an early and reliably predictive marker for AIDS disease progression and reflects pathogenicity. This parameter can be easily assessed in study protocols for drug and vaccine tests in monkeys as in man.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test. peerReviewed

Various simian immunodeficiency virus (SIV)sm/mac and simian/human immunodeficiency virus (SHIV) strains are used in different macaque species to study AIDS pathogenesis, as well as to evaluate candidate vaccine and anti-retroviral drugs efficacy. In this study we investigated the effect of route of infection, species of macaques and nature of virus stock on early plasma viral RNA load. We monitored the plasma RNA concentrations of 63 rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) infected with well-characterised virus stocks administered either by oral, rectal, vaginal or intravenous (i.v.) routes. In SIV(mac)-infected macaques, no significant difference in plasma RNA loads was observed between the rectal, oral and i.v. routes of infection. Cynomolgus macaques developed lower steady state SIV plasma RNA concentrations compared with rhesus macaques and no significant difference was observed between rectal and i.v. routes of infection. In SHIV(89.6p)-infected macaques, no difference between species or between route of infection was observed with this particular chimeric virus.

During the course of simian immunodeficiency virus (SIV) infection, nearly 15% of rhesus macaques (Macaca mulatta) and up to 40% of cynomolgus macaques (Macaca fascicularis) developed SIV-associated non-Hodgkin's lymphomas. Most of these malignant lymphomas harbored lymphocryptoviruses, which are closely related to the human Epstein-Barr virus (EBV; Herpesvirus M. mulatta and Herpesvirus M. fascicularis). To characterize the oncogenic role of simian EBV infection for lymphomagenesis during SIV infection, expression of the EBV-encoded latent membrane protein-1 (LMP-1) was analyzed in malignant lymphomas of SIV-infected rhesus macaques. Nine seropositive rhesus macaques suffering from B-cell lymphomas during the late phase of SIV infection were euthanized. Latency stages of EBV infection within malignant lymphomas and simian EBV-infected lymphoblastoid cell lines (LCL8664, H50) were characterized by analyzing expression of the EBV-encoded nuclear antigens EBNA-1, EBNA-2, and small RNAs EBER1/2. In parallel, the presence of viral LMP-1 transcripts was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Results were compared with findings in AIDS-associated malignant lymphomas in two patients infected with human immunodeficiency virus (HIV)-1. Rhesus macaques developed high-grade B-cell lymphomas of the centroblastic (five of nine), immunoblastic (two of nine), centroblastic-centrocytic (one of nine), and Burkitt-like (one of nine) subtypes within 18-29 months postinfection with SIV(mac)251/32H. The presence of Herpesvirus M. mulatta was detected in eight of nine cases. Transcription of the viral oncogene LMP-1 could be demonstrated within the simian EBV-infected cell lines as well as in four of nine SIV-associated malignant lymphomas. These four cases and both of the HIV-1-related non-Hodgkin's lymphomas expressed the full spectrum of latent EBV gene products (LMP-1, EBER1/2, EBNA-1, EBNA-2) and were thus classified as latency type III stages of EBV infection. Simian EBV infection was demonstrated in 90% of lymphomas in SIV-infected rhesus macaques. Analysis of LMP-1 expression suggests an important role for this viral oncogene in the pathogenesis of both SIV and HIV-1-associated malignant lymphomas.

Losing the decisive virus-specific functions of both CD4(+) and CD8(+) T lymphocytes in the first weeks after immunodeficiency virus infection ultimately leads to AIDS. The SIV/rhesus monkey model for AIDS was used to demonstrate that a 4-week chemotherapeutic reduction of viral load during acute SIV infection of macaques allowed the development of a competent immune response able to control virus replication after discontinuation of treatment in two of five monkeys. Increasing SIV-specific CD4(+) T-helper-cell proliferation was found in all macaques several weeks after treatment, independent of their viral load. However, only macaques with low viral loads showed persistent T-cell reactivity of lymph node cells. In contrast to animals with higher viral loads, T-helper-cell counts and memory T-helper cells did not decline in the two macaques controlling viral replication. Lymphocyte apoptosis was consistently low in all treated macaques. In contrast, high CD8(+) lymphocyte death but only slightly increased CD4(+) lymphocyte apoptosis were observed during the first weeks after infection in untreated control animals, indicating that early apoptotic death of virus-specific CTL could be an important factor for disease development. Antiretroviral treatment early after infection obviously retained virus-specific and competent T lymphocytes, whereby a virus-specific immune response could develop in two animals able to control the viral replication after cessation of treatment.

Mitochondrial genes overexpressed in human and monkey B-cell non-Hodgkin lymphomas (B-NHLs) were sought via subtraction hybridization, cloning, and differential screening of the resulting cDNA libraries. The cDNAs of mitochondrial genes constituted an appreciable proportion of all lymphoma-specific cDNAs. Lymphomogenesis was associated with upregulation of a set of mitochondrial genes, which varied with lymphoma type but always included NADHIV. A possible association between upregulation of certain mitochondrial genes and cell malignant transformation is discussed.

Recombinantly expressed VP1-virus-like particles (VP1-VLP) of human polyomavirus JC virus (JCV) were described recently as a new DNA transporter system. It was shown that DNA molecules could be packaged into VP1-VLP during a controlled chemical reassociation/dissociation process. In the present study VP1-VLP were studied as carriers for pharmaceutical substances. Propidium iodide (PI) was packaged into VP1-VLP as a reporter molecule. The PI-containing VP1-VLP could be detected directly by flow cytometry. The fluorescence intensity of the VP1-VLP depended strongly on the initial PI concentration. This packaging method is easy to handle and applicable to viruses and VP1-VLP which can be dissociated and reassociated chemically.

Infection with SIVmac251 in some rhesus monkeys (Macaca mulatta) leads to B-cell non-Hodgkin's lymphomas (B-NHL) clinically similar to that of HIV-infected AIDS patients. To further characterize the SIV-associated B-NHL we have generated genetic profiles of malignant cells by subtractive hybridization and Northern blot analysis. We have analyzed 21 clones of a subtracted cDNA library corresponding to overexpressed genes in diffuse large B-cell (DLBCL) SIV-associated monkey lymphoma. Eight of these clones represent a sequence homologous to an abundant transcript from KG-1 cells originally established from a human myelogenous leukemia. The protein encoded has a 60% similarity to a hypothetical glycine-rich transmembrane signal protein of Caenorhabditis elegans and 25% similarity to the ret finger protein. The other cDNA clones contained sequences of the serum amyloid A gene (SAA), the alpha1-acid glycoprotein gene (AGP), the ribosomal protein S3a (RPS3a) and L8 (RPL8) genes, the interferon-inducible gene (INF-ind), the metastasin gene (mts1), and the NADH dehydrogenase I gene (ND-I). The remaining cDNA clones consisted of yet unknown sequences. In addition, we detected an up-regulation of the cytochrome c oxidase II gene (COX-II), the ND-IV gene, and the SET oncogene by Northern blot hybridization in three SIV-associated NHLs of different histomorphological classification. All these genes have not previously been found to be overexpressed in B-NHL.

The early events during infection with an immunodeficiency virus were followed by application of pathogenic simian immunodeficiency virus atraumatically to the tonsils of macaques. Analyses by virologic assays and in situ hybridization revealed that the infection started locally in the tonsils, a mucosal-associated lymphoid organ, and quickly spread to other lymphoid tissues. At day 3, there were few infected cells, but then the number increased rapidly, reaching a high plateau between days 4 and 7. The infection was not detected in the dendritic cell–rich squamous epithelium to which the virus was applied; instead, it was primarily in CD4 + tonsillar T cells, close to the specialized antigen-transporting epithelium of the tonsillar crypts. Transport of the virus and immune-activating stimuli across this epithelium would allow mucosal lymphoid tissue to function in the atraumatic transmission of immunodeficiency viruses.

We tested the possibility that lymphocytes and serum obtained directly from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) induce infection in rats. Inbred Fischer F344 immunosuppressed rats were inoculated intravenously with 10x10(6) peripheral blood mononuclear cells (PBMC; 3 rats) and serum (3 rats) obtained from a HAM/TSP patient, who was seropositive and polymerase chain reaction (PCR)-positive for the HTLV-I proviral genome. Antibodies to HTLV-I appeared in the rat sera 2 months later; rat peripheral blood lymphocytes, spleen, salivary gland, and spinal cord were found to contain the proviral genome. Control rats inoculated with normal donor PBMC and serum tested negative for the HTLV-I antibodies and for the HTLV-I proviral genome by PCR. The positive control F344 rats inoculated with 5x10(6) cells of a SLB-1 HTLV-I cell line were found to be infected after 2 months. This study demonstrates for the first time that HTLV-I can be transmitted not only by human cellular components but also by human cell-free sera in a rat model.

Increased rates of T-cell apoptosis have been detected in human immunodeficiency virus (HIV)-infected individuals and in the simian immunodeficiency model (SIV) for AIDS research. We have infected macaques with virulent SIV or SIV/HIV hybrid viruses (SHIV) of different pathogenic potentials to study the early kinetics of apoptosis in this model. Animals infected with SIV showed an increased degree of apoptosis in their peripheral blood mononuclear cells as early as 8 weeks after virus inoculation. Apoptotic cells were detected in the CD4 and CD8 cell populations of infected animals. In contrast, apathogenic SHIV did not lead to increased lymphocyte apoptosis and moderately pathogenic SHIV induced only transient apoptosis. T-cell death was temporally linked to viral replicationin vivo.Furthermore, lymphocyte apoptosis in infected macaques was associated with impaired proliferative responses of helper T-cells and with CD4 cell depletion. The monkey model described here provides the opportunity for testing early therapeutic interventions to prevent virus-induced programmed cell death and the subsequent onset of AIDS.

A high prevalence of human T-lymphotropic virus type I (HTLV-I) infection among Israeli Jews was previously reported. In the present study, screening for HTLV-I of Israeli Jews was expanded to 10 ethnic groups. HTLV-I antibodies were tested by the particle agglutination assay, ELISA, and by Western blot as a confirmatory method. The HTLV-I proviral genome was tested by nested PCR with tax primers (SK43/SK44 and Tr101/Tr102). The PCR tests were carried out in all seropositive subjects and the seronegative family members of the seropositives subjects in the Iranian population. Sixty-eight of the 1,679 subjects (4.1%) were found to be seropositive. The Jews originating from Mashhad had the highest infection rate of 60/306 (20%). Of the 479 Iranian non-Mashhadi Jews, 6 (1.3%) were seropositive. Of the 894 non-Iranian Israelis, only 2 (0.2%) were seropositive. HTLV-I proviral DNA was found in the peripheral blood lymphocytes of 66 out of 68 seropositive subjects and 6 out of 75 seronegative subjects. Sixty out of 123 (49%) Mashhadi Jews and 8 out of 14 (57%) non-Mashhadi Iranian Jews were PCR-positive. Three out of three seropositive non-Iranian Israelis were PCR positive. One non-Iranian Israeli (who originated from Ukraine) without family connections to the Iranian Jews was also PCR-positive. One hundred eighteen saliva samples (84 from subjects of Mashhadi origin, 31 from Iranian origin, and 4 of other origins) were also screened. Antibodies for HTLV-I were found in 23 out of 46 saliva samples from the individuals with particle agglutination (PA) and/or PCR-positive findings in blood. Twenty out of 23 PA-positive saliva samples also contained the proviral DNA. It is concluded that HTLV-I infection in Israel is mainly limited to Jews originating from Iran (most of them from Mashhad) and their family members.

The updated results of current pathomorphological investigations in SIV-infected rhesus monkeys (Macaca mulatta) are summarized. After experimental infection with several SIVmac251 subtypes and various vaccination trails, 147 rhesus monkeys were morphologically examined until now. The pathology of the gastrointestinal tract in SIV-infected animals resembled those of human cases with HIV and AIDS. Alterations were considered to be primary SIV-induced (SIV enteropathy, giant cell disease) or secondary caused by opportunistic agents. Typical secondary gastrointestinal opportunistic infectious agents were parasites (Cryptosporidium sp., Trichuris sp., Trichomonas sp., Spironucleus sp.), viruses (cytomegalovirus, adenovirus) and bacteria (Mycobacterium simiae). Five animals developed malignant lymphomas involving the intestinal tract. The present observations revealed that SIV infection of rhesus monkeys provide an excellent model for studies on the pathogenesis of HIV in man.

Live-attenuated simian immunodeficiency virus (SIV) protects macaques against challenge with pathogenic SIV. To evaluate the safety of such vaccines, an investigation of whether or not nef-deleted SIV could be reactivated in vivo by immune activation of the host was conducted. In addition, monkeys infected with apathogenic SIV/HIV-1 chimeric viruses, and two control monkeys that had suppressed replication of pathogenic SIV were examined. During the infection virus became undetectable or persisted at a low level of replication in all monkeys. At this time-point 11 monkeys were immune-activated by a vaccinia virus (VV) superinfection. After VV infection up to 80% of their lymphocytes showed expression of the activation markers CD25 and CD69 over 2 weeks. However, only the two non-progressing monkeys infected with pathogenic SIV showed a noticeable but transient enhancement of SIV replication and increased SIV antibody titres. By contrast, in monkeys infected with apathogenic immunodeficiency viruses no change in virus load was observed. Therefore, attenuated immunodeficiency viruses cannot be reactivated in vivo by a VV-induced immune activation.

We have assessed the diagnostic efficacy of a novel polyclonal rabbit antiserum directed to the recombinant major capsid protein VP1 of JC virus (JCV). Immunohistochemistry for VP1 was compared to non-radioactive JCV DNA in situ hybridization (ISH) in ten cases of progressive multifocal leukoencephalopathy (PML). Tissue sections from postmortem brains were studied from PML patients suffering from immunodeficient conditions of various causes: immunodeficiency syndrome (AIDS, n = 7), severe combined immune deficiency due to adenosine deaminase deficiency (n = 1), sarcoidosis (n = 1) and leukemia (n = 1). VP1 immunohistochemistry demonstrated the presence of JCV in lesional oligodendrocytes of all PML patients, whereas ISH was able to detect JCV in nine out of ten cases. We conclude that VP1 immunohistochemistry is a specific, sensitive and rapid method for confirming the diagnosis of PML.

SIV infection of macaques is a well-established animal model for studying the pathogenesis of HIV infection in humans. During the course of SIV infection, up to 40% of cynomolgus macaques (Macaca fascicularis) develop SIV-associated non-Hodgkin's lymphomas. In the present study, we characterized malignant lymphomas of SIV mac 251/132H-infected rhesus macaques (Macaca mulatta) of our cohort in terms of clinical outcome, histopathology and EBV association. Histopathologic changes of lymphoid malignancies were classified according to the Kiel classification. For detection of the EBV-encoded small RNAs EBERI and EBER2, a method of non-isotopic in situ hybridization was established. The presence of EBNA-2 antigens was assessed by immunohisto-chemistry. Seven of 43 rhesus macaques developed highly malignant B-cell lymphomas of the centroblastic, immunoblastic and Burkitt subtypes within 18-29 months post-experimental SIV infection. In situ hybridization revealed the presence of small EBERI and -2 RNAs in 6 of 7 disease cases. EBNA-2 antigens could be demonstrated in only 4 of 7 tissue specimens. As expected, the Burkitt-type of lymphoma was negative for EBNA-2 antigen staining. In accordance with findings on SIV-associated lymphomas of cynomolgus macaques, infection with an EBV-related herpesvirus could be demonstrated in almost 90% of lymphomas in SIV-infected rhesus macaques. In contrast, the presence of EBV in lymphomas had been documented previously in only 30-40% of HIV-infected patients. Further studies should thus define the precise role of herpesvirus infection for lymphomagenesis in SIV- and HIV-induced immunodeficiency.

Herpes virus saimiri(HVS) transforms human T-cellsin vitroto stable growth. These T-cell lines retain their immunological characteristics of the parent cells and do not release infectious virus. Recently, lymphocytes of Old World monkeys were efficiently transformed by HVS. In parallel to these studies we initiated transformation experiments by infecting peripheral blood cell cultures of 45 monkeys, 35 rhesus and 10 cynomolgus macaques. In only three cases, we obtained transformed T-cell lines. The transformed T-cells were largely double-positive for CD4 and CD8. They responded with increased proliferation to mitogenic or IL-2 stimulation and transcribed mRNA for IL-2, IL-4, and IL-10. However, most initiated T-cell cultures from macaques developed giant cells. The cytopathic agent was identified assimian foamy virus(SFV) as confirmed by PCR, immunofluorescence, and coculture experiments. Treatment of the T-cell cultures with AZT- and SFV-specific sera did only shortly prolong the life-span of the cultures. Therefore, the reactivation of SFV caused remarkable difficulties in the establishment of macaque T-cell lines by HVS. This seems to be a general problem since most animals from several breeding colonies are SFV-positive.

Altered cytokine transcription might play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection in humans. The infection of rhesus macaques with simian immunodeficiency virus (SIV) provides a relevant animal model for HIV infection. Therefore, we evaluated the cyokine transcription of phytohemagglutinin (PHA)-stimulated lymphocytes in the early phase after infection of four rhesus macaques with pathogenic SIV-mac239. To determine transcription of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6, and IL-10 we established a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). After inoculation with SIV, all monkeys became productively infected and developed an acquired immunodeficiency syndrome (AIDS) like disease. Infection was associated with a proliferation dysfunction of monkey lymphocytes in response to PHA. In addition, a decreasing overall cytokine transcription could be observed during the course of SIV infection. These findings demonstrate that an impairment of the lymphocyte function is associated with a reduced cytokine transcription in the early phase of an immunodeficiency virus infection. The observed differences of cytokine expression might contribute to the impaired immune response of SIV-infected monkeys and HIV-infected humans.

A new in vitro system for the production of the human polyomavirus JC virus (JCV) was established to circumvent the need for virus growth in primary human fetal glial cells (PHFG). The permanent cell line SVG, transformed by an origin-defective mutant of Simian Virus 40 (SV40) was used to grow JCV. JCV-specific RNA could be detected at day 5 and viral antigen at day 6 post infection (p.i.). Virus production peaked at day 16. Virus could be purified by differential centrifugation. The purified fraction consisted mainly of mature particles but contained also pentamers of the major structural virus protein 1 (VP1). The VP1-pentamers could be purified to near homogeneity. The purified virus particles stimulated a specific T-cell proliferation of peripheral blood monocytes (PBMCs) of a patient with progressive multifocal leukoencephalopathy (PML) and of two healthy individuals. In addition, JCV-particles and VP1-pentamers reacted specifically in an ELISA with a series of five PML-patient sera and four sera of individuals not affected by PML. These results demonstrate that purified whole virus particles are suitable for the analysis of specific cellular and humoral immune responses to JCV.

Prion diseases are neurodegenerative disorders, affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). To generate monospecific antisera against human prion proteins we have immunized mice with DNA coding for different human prion proteins. We constructed immunization vectors expressing individual genotypes of either the cellular prion gene (PRNP) or mutant forms under appropriate promoters. This approach avoids the preparation of infectious material for immunization. To circumvent immunological tolerance prion protein-deficient PrP00 mice were used for the DNA-mediated immunization. Thereby monospecific sera were raised capable of specifically precipitating in vitro synthesized human prion proteins. With prion protein-specific peptide ELISAs, we found that antibodies are predominantly directed against the octapeptide repeat region and to a lesser extent to regions comprising the signal peptide, the neurotoxic domain or the GPI anchor. In contrast, prion gene-positive (PrP+/+) BALB/c mice immunized under the same experimental conditions as the PrP00 mice did not respond with antibody formation against the human prion protein. This is the first report clearly showing that immune competent prion protein-deficient mice react with a vigorous polyclonal immune response after DNA-mediated immunization with human prion gene sequences.

Summary Background The GB virus C (GBV-C) and the hepatitis G virus (HGV) have been detected in patients with acute indeterminant hepatitis and post-transfusion hepatitis. However, the role of the new hepatitis viruses in the aetiology of fulminant hepatitis is little understood. We investigated the presence of GBV-C/HGV in patients with fulminant hepatic failure. Methods Serum samples from 22 German patients with fulminant hepatic failure and 106 symptom-free blood donors (controls) were studied for presence of GBV-C RNA by seminested reverse transcriptase PCR. Primer sequences were derived from the published gene sequences of the conserved NS3 region of the GBV-C prototype and the published isolates. Nucleotide and aminoacid sequences of GBV-C-positive isolates, the control RNA, and the published HGV and GBV-C prototype sequences were compared by multiple sequence alignment. We also compared the GBV-C sequences of virus-positive patients who had fulminant hepatic failure with those of 19 patients with chronic hepatitis from our centre. In addition, we searched databases and published papers for further GBV-C helicase sequences in patients with non-fulminant hepatitis. Findings GBV-C RNA was detected in 11 (50%) of the 22 patients with fulminant hepatic failure and in five (4·7%) of 106 control-group blood donors. Among the patients with fulminant hepatic failure, six of seven with fulminant hepatitis B and five of ten with fulminant non-A-E hepatitis were positive for GBV-C RNA. Analysis of nucleic acid sequences showed six mutations at defined positions in all 11 patients with fulminant hepatic failure who were positive for GBV-C. None of these mutations were found in the five GBV-C-positive control-group blood donors. Of the six nucleotide changes, four caused no aminoacid changes, whereas two mutations at position 100 (G to T) and 102 (T to C) led to an alanine to serine change in the predicted translation product. However, comparison with GBV-C sequences of patients with non-fulminant hepatitis showed that this aminoacid mutation was not specific for fulminant hepatic failure. The sequence-motif containing the six nucleotide mutations detected in all patients with fulminant hepatic failure was found in only two of 19 German patients with chronic hepatitis from our centre, and in only one of 88 GBV-C sequences from non-fulminant patients reported by others. Interpretation The frequency of GBV-C RNA is higher in fulminant hepatic failure than in any other group of patients with hepatitis, particularly in patients with fulminant hepatitis B or fulminant non-A-E hepatitis. A specific strain of GBV-C may occur in serum of German patients with fulminant hepatic failure.

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.

Objective : To examine the biological properties of HIV-1/SIV mac chimeric viruses from HIV-1 isolates that have different replication rates, cell tropisms and cytopathicities. Design and methods : Four chimeric viruses with gag, pol, vif, vpx, nef and long terminal repeats of SIV mac and vpr, tat, rev, vpu and env of various HIV-1 isolates were constructed and compared in vitro. Cynomolgus monkeys were inoculated with two chimeras that were replicative in monkey peripheral blood mononuclear cells (PBMC). Results : The type-specific neutralization of the chimeras by monoclonal antibodies 0.5β and μ5.5, which recognize V3 of HIV-1 RIB and HIV-1 MN respectively, was observed to be similar to those of the parental viruses, HIV-1 NL432 , HIV-1 HAN2 and HIV-1 SF13 . The chimeras constructed from HIV-1 SF2 and HIV-1 SF13 , which were isolates from the same individual but from different disease stages, reflected their parental properties, that is, the isolate from the later stage was rapid-high replicating, was more cytopathic and had a wider host range. Chimeras constructed from HIV-1 HAN2 , HIV-1 SF13 and HIV-1 NL432 were infectious to macaque monkeys, although the monkeys infected with the chimera from HIV-1 SF13 showed lower virus loads and shorter viremic periods than those infected with the others. Conclusions : Chimeras have in vitro properties that are similar to those of their parental HIV-1 isolates, but their growth in macaque PBMC was dependent on which HIV-1 isolate was used. Evaluation of a vaccine by challenging with viruses possessing different antigenicities has become possible in macaque monkeys using newly constructed chimeras.

The efficacy of three SIVmac32H gp130 vaccines was compared in rhesus monkeys. Three rhesus monkeys were each immunized over a period of 20 weeks with a total of 600 microgram virion-derived gp130 oligomers (O-gp130) mixed with keyhole limpet hemocyanin and emulsified with incomplete Freund's adjuvant. Three other monkeys were infected with 5 x 10(8) PFU of vaccinia virus wild type (VV-wt) while three additional animals received an equivalent dose of VV expressing the gp130 of SIVmac (VV-gp130). At Week 8, the two VV-wt animals received an additional immunization with 100 microgram O-gp130 each. All VV-infected animals then received booster immunizations at Weeks 12, 16, and 20 with a total of 300 microgram O-gp130 per animal. All animals along with two controls were challenged iv with 50 MID50 of T-cell-grown SIVmac32H at Week 22. Four weeks after the challenge and thereafter, both controls and one animal from either VV group were infected as demonstrated by polymerase chain reaction (PCR), virus isolation, and antibody response. In contrast, all O-gp130 animals and one animal each from the VV-wt and the VV-gp130 group were completely protected as shown by negative PCR and virus reisolation. One animal of the VV-gp130 group was partially protected, since it remained virus isolation negative but became PCR positive. All protected animals did not develop a secondary antibody response. Six months after the first challenge, the five completely protected animals were reimmunized twice 4 weeks apart with a total of 200 microgram O-gp130 per animal. Two weeks later, all animals were challenged with 5 MID50 of the SIVmac32H/spI prepared from the spleen of an immunized, but unprotected SIV-infected rhesus monkey. After the second challenge, all three control animals and one of the vaccinees become productively infected. In contrast, two animals were completely protected, one from the former O-gp130 and one from the former VV-gp130 group. One animal from the former VV-wt group was only DNA-PCR positive and thus partially protected. Therefore, immunization with virion-derived gp130 oligomers of SIVmac32H can confer protection against the infection with T-cell-grown SIVmac32H as well as the ex vivo isolate SIVmac32H/spI.

To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as B p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli -expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.

A chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu, env, and nef genes of human immunodeficiency virus type 1 was generated. The chimeric virus, NM-3n, grew competently in peripheral blood mononuclear cells from cynomolgus monkeys like the parental SIVmac. Two cynomolgus monkeys and one rhesus monkey inoculated with NM-3n raised antibodies to SIVmac Gag and HIV-1 Env. The antibodies raised in the cynomolgus monkeys persisted for at least 1.7 years. The antibodies contained virus neutralizing activity not only to the original chimeric virus but also to the parental HIV-1. Infectious viruses were isolated from one of the cynomolgus monkeys 37 and 63 weeks after inoculation and from the rhesus monkey continuously from 6 weeks after infection onward. The recovered virus maintained its chimeric structure but included several clones with mutations in the env V3 region. When the recovered virus was inoculated to another rhesus monkey, no difference in the frequency of virus recovery was seen from the originally infected monkeys. These carrier monkeys have so far shown no sign of the disease.

The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.

The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.

The purpose of this study was to characterize antigenic determinants on structural polypeptides of human immunodeficiency virus type 2 (HIV-2ben). Therefore, three HIV-2-specific monoclonal antibodies (mAbs) against the p24 core protein (gag) and one mAb against the gp130 envelope glycoprotein (env) were produced. In addition to p24 the anti-core mAbs recognized the primary translation product of the viral gag gene p55 and an intermediate cleavage product p41. Core mAbs cross-reacted with another HIV-2 isolate (HIV-2rod), and several simian immunodeficiency viruses (SIVagm TYO7 and SIVmac), but not with SIVmnd and the HIV-1 isolates investigated (HIV-1han and HIV-1lai). The env mAb cross-reacted with HIV-2rod and SIVmac but not with SIVagm, SIVmnd or HIV-1. In competition assays and with epitope mapping possible binding sites for the mAbs were identified. The processing of HIV-2 core proteins is compared in retrovirus-infected T cell lines and during the expression by recombinant vaccinia virus. Finally, the mAb XIV DC10 which recognized a highly conserved epitope could be useful for an assay to detect HIV-1 and HIV-2 simultaneously. II D8 is the first mAb raised against HIV-2 env glycoprotein.

Five monoclonal antibodies (mabs) specific for the envelope proteins of a simian immunodeficiency virus of African green monkeys (SIVagm) have been raised. Two mabs were directed against distinct epitopes on the transmembrane protein gp41. A conformational epitope on the gp130 was recognized by three mabs. This is the first report on mabs specific for SIVagm-gp130. Studies of the cross-reactivities revealed that the epitopes recognized by the env-directed mabs are conserved species-specifically in SIVagm isolates. Therefore, these mabs can be used to distinguish SIVagm strains from other virus groups.

The external envelope glycoprotein gp130 of a simian immunodeficiency virus isolated from an African green monkey (SIVagmTYO-7) was purified as micellar complexes. The molecular weight of the gp130 micelles was about 700 K. On electron microscopy, the micelles appeared as spherical particles with a diameter of 15 to 20 nm. Such aggregates consisted of about 4 to 5 gp130 monomers. Hyperimmune sera raised in rabbits and rhesus monkeys against these gp130 micelles exhibited titers between 10 5 and 10 6 . Such sera inhibit the CD4 binding of gp130 and neutralize SIVagmTYO-7 and SIVmac251 but not HIV-2ben.