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BACKGROUND Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti–infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14–CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

BACKGROUND: When xenotropic murine leukemia virus–related virus (XMRV) was first reported in association with chronic fatigue syndrome, it was suggested that it might offer a risk to blood safety. Thus, the prevalence of the virus among blood donors and, if present, its transmissibility by transfusion need to be defined. STUDY DESIGN AND METHODS: Two populations of routine blood donor samples (1435 and 13,399) were obtained for prevalence evaluations; samples from a linked donor-recipient repository were also evaluated. Samples were tested for the presence of antibodies to XMRV-related recombinant antigens and/or for XMRV RNA, using validated, high-throughput systems. RESULTS: The presence of antibodies to XMRV could not be confirmed among a total of 17,249 blood donors or recipients (0%; 95% confidence interval [CI], 0%-0.017%); 1763 tested samples were nonreactive for XMRV RNA (0%; 95% CI, 0%-0.17%). Evidence of infection was absent from 109 recipients and 830 evaluable blood samples tested after transfusion of a total of 3741 blood components. CONCLUSIONS: XMRV and related murine leukemia virus (MLV) markers are not present among a large population of blood donors and evidence of transfusion transmission could not be detected. Thus, these viruses do not currently pose a threat to blood recipient safety and further actions relating to XMRV and MLV are not justified.

BACKGROUND: Xenotropic murine leukemia virus–related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection. STUDY DESIGN AND METHODS: Plasma from 1000 US, 100 human immunodeficiency virus Type 1–infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction. RESULTS: XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I–infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I–infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal. CONCLUSIONS: Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

The diagnosis of chronic Chagas' disease is generally made by detecting antibodies to Trypanosoma cruzi . Most conventional serological tests are based on lysates of whole parasites or semipurified antigen fractions from T. cruzi epimastigotes grown in culture. The occurrence of inconclusive and false-positive results has been a persistent problem with the conventional assays, and there is no universally accepted gold standard for confirmation of positive test results. We describe here an immunoblot assay for detecting antibodies to T. cruzi in which four chimeric recombinant antigens (rAgs), designated FP3, FP6, FP10, and TcF, are used as target antigens. Each of these rAgs is composed of several antigenically distinct regions and includes repetitive as well as nonrepetitive sequences. Each rAg is coated as a discrete line on a nitrocellulose strip. Assay sensitivity was assessed by testing 345 specimens known to be positive for antibodies to T. cruzi . All 345 of these samples showed two to four reactive test bands in addition to the three on-board control bands that are on each strip. Assay specificity was determined by testing 500 specimens from random U.S. blood donors, all of which gave negative results. Based on the results obtained in this study, we propose the following scheme for interpretation of test results: (i) no bands or a single test band = a negative result; (ii) two or more test bands with at least one band showing intensity of 1+ or higher = a positive result; and (iii) multiple faint test bands (±) = indeterminate result. Based on this scheme, the prototype immunoblot assay showed sensitivity of 100% ( n = 345) and specificity of 100% ( n = 500). Additionally, all 269 potentially cross-reacting and T. cruzi antibody-negative specimens tested negative in our immunoblot assay. The rAg-based immunoblot assay has potential as a supplemental test for confirming the presence of antibodies to T. cruzi in blood specimens and for identifying false-positive results obtained with other assays.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that can be transmitted by transfusion. The diagnosis of chronic T. cruzi infection is generally made by detecting specific antibodies that bind to parasite antigens. The aim of this study was to assess the sensitivity and specificity of a new serologic assay for antibodies to T. cruzi on a fully automated analyzer (PRISM, Abbott Laboratories). STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay based on chimeric recombinant antigens and run on the automated PRISM system was developed for detecting antibodies to T. cruzi in human serum and plasma. Assay specificity was evaluated by testing samples from random blood donors and from a diverse group of specimens from persons with diseases or conditions often associated with false-positive reactions in T. cruzi assays. Sensitivity was determined by testing 377 geographically diverse T. cruzi antibody–positive specimens. RESULTS: Six of 7911 samples (0.08%) from random donors were repeatedly reactive in the prototype PRISM Chagas assay. One of these was reactive in three other tests, including the radioimmune precipitation assay and was presumed to be a true positive. Hence, the specificity was 99.94 percent (7905/7910) in the negative donor group studied. All 377 T. cruzi antibody–positive specimens were positive in the prototype assay and thus the sensitivity was 100 percent. CONCLUSION: The results obtained to date, in terms of sensitivity as well as specificity, strongly suggest that the PRISM Chagas assay should function well as a tool for screening blood for serologic evidence of T. cruzi infection.

HIV-1 is classified into three groups, M (major), N (non-M non-O), and O (outlier); each group arose from a separate transmission of SIVcpz into humans. HIV-1 group N was recently discovered and infections with this virus are rare with only eight documented cases. All group N infections have been found in Cameroon and there is no evidence of direct linkage between the infected patients. We report here the identification of HIV-1 group N infections in a husband and wife. The group N infection in the husband, 1131-03, was identified first based on seroreactivity in peptide EIAs and confirmed by PCR amplification of group N viral sequences. Subsequently the wife, 1015-04, was evaluated and confirmed to also be infected with a group N virus. Near full-length viral genomes were amplified and sequenced from each patient's specimen. The low level of diversity between the two viral sequences provides evidence of horizontal transmission of group N from one spouse to the other. Patient 1131-03 was receiving antiviral therapy consisting of reverse transcriptase inhibitors; the treatment appears effective for suppression of group N viral replication based on apparently low viral load in plasma specimens collected from the patient and the absence of drug resistance mutations in RT sequences amplified from 1131-03. This report brings to 10 the number of group N infections identified and to 5 the number of group N genomes sequenced. Although group N infections continue to be rare, group N is a pathogenic virus and its prevalence needs to be monitored.

Over the past 16 years, the epidemics of HIV infection and AIDS have created many challenges for those working to develop serologic and genetic tests to diagnose infection, antiviral agents to treat infection, and vaccines to prevent infection. Perhaps the greatest challenge comes from the realization that HIV is not a single virus, but a group of related viruses. The remarkable genetic heterogeneity of HIV enables certain HIV strains to elude detection by some of our most widely used serologic assays, to develop resistance to antiviral compounds within weeks to months, and to escape from immune responses generated by natural infection and immunization. This article discusses the range of HIV genetic variation and possible reasons for this variation, and focuses on a specific subset of HIV–1 viruses known as group O .

Serologic reactivities of serum or plasma from 55 HIV-1 subjects in four countries—Brazil, Rwanda, Thailand, and Uganda—were examined by V3 peptide immunoassay. Forty-seven (85.5%) of the 55 specimens tested positive to the homologous peptide. A strong correlation between serotype (i.e., pattern of serologic reactivity with a panel of peptides) and genotype was not found. However, the V3 peptide immunoassays may be useful for epidemiologic studies to trace the distinctive HIV-1 strains from different geographic regions of the world. The serology data obtained may be useful for the development of effective V3-based vaccines.

HIV-1 and HIV-2 isolates representing various geographic regions and distinct viral subtypes were examined for their ability to establish both in vitro and in vivo productive infections of Macaca nemestrina (pigtail macaque) peripheral blood mononuclear cells.Animals were inoculated with either autologous cell-associated or cell-free viral preparations of selected isolates. HIV-specific immune responsiveness, hematologic changes, genetic variation, and virus burden were monitored as delineators of HIV pathogenesis.HIV-2 replication in vitro and in vivo correlated with nascent antigen production and rising viral titers as determined by infectious center assays. Infection was detectable by polymerase chain reaction amplification of proviral sequences in macaque cells as early as 1 week postinoculation. Two distinct patterns of CD4+ cell depletion induced by HIV-2 infection were observed during the first month postinoculation and characterized by a moderate loss sustained through 20 weeks postinoculation or a substantial loss maintained long-term (90 weeks). Identity between inoculating viral stocks and subsequent viral isolates from animals was established comparatively by limited sequence analysis of specific domains within the HIV-2 pol and env genes. In contrast, replication of HIV-1 isolates was limited or only semipermissive in vitro. Intravenous inoculation of HIV-1 field isolates, using conditions successful for HIV-2 (for example, identical viral titers), failed to establish a productive viral infection leading to seroconversion of fluctuations in hematologic cell markers. Infection with a high-titer inoculum of a laboratory-adapted HIV-1 strain in vivo, as demonstrated by polymerase chain reaction analysis, produced seroconversion in the absence of overt viral replication or hematologic variations in one out of four animals.This system provides for multifaceted modeling of HIV pathogenesis, primarily with HIV-2 and potentially with HIV-1/-2 chimerics, in support of immunotherapeutic developments and critical evaluation of intervention practices.

Xenotropic Murine Leukemia Virus-related Retrovirus (XMRV) was identified from prostate cancer tissue using DNA based ViroChip technology as well as in a cohort of chronic fatigue syndrome patients. To delineate the infection dynamics and dissemination in vivo, we have thus far infected 7 healthy rhesus macaques and 2 pigtailed macaques. Results show that XMRV induces a chronic and clinically silent infection that is nevertheless persistent and susceptible to reactivation in vivo. While XMRV seems rapidly cleared from the blood circulation in healthy macaques, XMRV protein positive CD4+ T cells were detected in all lymphoid organs throughout infection. However, among all organs subjected to in situ detection, mucosal sites overall and sexual organs showed markedly higher frequencies of XMRVgag positive cells, including gastrointestinal mucosa, pulmonary environment and organs from the reproductive tract. Of interest, the lineage of cells that were XMRV positive markedly differed among the different sites, including CD4+ T cells in the GI mucosa, alveolar macrophages in the lung, epithelial cells in the prostate, seminal gland, vagina and cervix and interstitial cells in the testes. The latter were consistently observed during acute and chronic infection, suggesting the potential for sexual transmission of XMRV. In fact, a single atraumatic mucosal exposure with a high dose of XMRV virus into the urethra resulted in infection of 1 out of 4 macaques providing proof of concept that such transmission is possible. However, additional work is needed to fully investigate potential modes of XMRV infection.

Background PCR-based testing has been widely utilized to assess the prevalence of Xenotropic Murine Leukemia Virus-related Virus (XMRV) in prostate cancer and chronic fatigue syndrome patients. An alternative approach, screening for antibodies elicited by XMRV infection, represents a more desirable option for large-scale epidemiologic studies. In this study, blood donor and retrovirus-infected populations were screened for serologic evidence of XMRV infection.

A simplified human immunodeficiency virus 1 (HIV-1)-specific IgA capture enzyme immunoassay (IgA-CEIA) was evaluated and compared with IgA-Western blot assay for early diagnosis of HIV-1 infection in infants born to seropositive women. A total of 232 coded sera collected prospectively from 70 infants were tested. All 25 sera from 10 HIV-1-negative infants born to seronegative mothers (negative controls) were negative by both assays. All 111 sera from 37 seroreverting, uninfected infants were negative by IgA-CEIA (specificity, 100%), whereas 110 of 111 sera were negative by IgA-Western blot assay (specificity, > 99%). Overall IgA-CEIA detected HIV-IgA in 20 (87%) of 23 infected infants, and IgA-Western blot assay detected HIV-IgA in 21 (91.3%) of 23 infants; specimen-wise agreement between the 2 assays was > 80%. Analysis of results by age group indicated that after 2 months of age both assays were equivalent with sensitivity ranging from 60 to 80%. Quantitative data provided by IgA-CEIA suggests that the bulk of HIV-1 IgA synthesis in most HIV-1-infected infants occurs after 2 months of age.

The licensure of combination human immunodeficiency virus type 1 and type 2 (HIV-1/HIV-2) enzyme immunoassays (EIAs) by the Food and Drug Administration has been accompanied by a recommendation that U.S. blood banks begin testing the nation's blood supply for HIV-2 by June 1, 1992. The performance of a recently licensed combination HIV-1/HIV-2 EIA (Genetic Systems) was evaluated using 3100 sera collected in the United States. A total of 2,049 sera were obtained from populations with low risk for HIV infections, and 1,051 sera from populations with high-risk behaviors. The combination EIA, in comparison with monospecific EIA, was found to be 100% sensitive for HIV-1 for both populations. The high-risk population had an HIV-1 seroprevalence rate of 17.4%, with a positive predictive value (PPV) of 97.3%. The low-risk population had an HIV-1 seroprevalence of 0.05% with a PPV of 8%. The incorporation of the combination EIA in various testing algorithms was also evaluated, and recommendations are given with consideration for the type of screening and populations involved.

Initial findings are reported on the genotype variation of HIV-1 in the Mara region of northwest Tanzania where the overall prevalence of HIV-1 was 7.3% during 1989-90. HIV-1 sequences from eight peripheral blood mononuclear cell specimens are reported. Seven specimens were collected in 1994 from residents of rural villages in the area while the eighth specimen was collected in 1987 from a person who originally lived in the Mara region moved to Kigoma Tanzania for several years then returned to the Mara region after developing AIDS. The genetic variability of the HIV-1 env Tanzania specimens is consistent with the pattern of increasing sequence divergence observed in that region of the world. The authors stress that sequence differences observed in comparison with older Tanzanian HIV specimens need to be followed up with greater numbers of sequences in order to determine the direction of change and eliminate sampling bias.

BACKGROUND: Reports that the human immunodeficiency virus type 1 (HIV- 1) group O variants are not reliably detected by some commercial diagnostic tests have raised concerns about the sensitivity of existing screening tests, especially with regard to blood safety. Although it is unlikely that these divergent strains are prevalent in North America, systematic, continuous surveillance is needed to monitor the potential spread of HIV variants into that region. STUDY DESIGN AND METHODS: Stored serum samples (n = 1072) from both high- and low-risk population groups at several sites in the United States and Puerto Rico were tested by peptide enzyme immunoassays specific for the prototypic HIV-1 group O strains, MVP5180 and ANT70. RESULTS: None of the 1072 samples examined had peptide reactivity that was consistent with HIV-1 group O infection. CONCLUSION: While no evidence of specific HIV-1 group O (MVP5180 or ANT70) infection was found in this study, the sensitivity of current tests has not been fully evaluated against the wide range of genetic variation of HIV. Therefore, it is important to continue active surveillance for HIV-1 and HIV type 2 strains, to characterize any divergent strains, and to judiciously modify tests to correct for any deficiencies in sensitivity.

HIV-1 strain diversity was examined in a study population that consisted of hospital and clinic patients from seven cities and villages located in the northwestern regions of Cameroon. Specimens were screened using a serological algorithm designed to identify HIV-1 group M, N, and O, and SIVcpz-like infections followed by RT-PCR amplification to characterize the infecting virus. The results show that the HIV epidemic in northwest Cameroon is dominated by HIV-1 group M CRF02_AG infections (57%). Additional group M subtypes present include A, D, F2, G, and CRF01_AE. Based on discordant subtype classification between gag and env sequences, a high percentage (23%) of viral strains appear to be unique intersubtype recombinants with the majority (88%) involving recombination with CRF02_AG. Group O prevalence is low accounting for only 0.4% of HIV infections. However, group O strain diversity is high; isolates from clades I, IV, and V, as well as unclassified and recombinant strains, were found. Three dual infections by HIV-1 group M and group O were identified and characterized. In two specimens, both group M and O sequences were amplified in gag, pol, and env suggesting the presence of both viruses. Analysis of the third specimen shows the presence of a group O virus and an intergroup M/O recombinant virus. Finally, no infections due to HIV-1 group N or SIVcpz-like strains were found in the study population.

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

The macaque monkey infected with simian immunodeficiency virus (SIV) is an animal model of the acquired immunodeficiency syndrome. We investigated a laboratory worker who was exposed by needlestick accident to blood from an SIV-infected macaque. Seroreactivity to SIV developed within 3 months of exposure, with antibody titres peaking from the third to the fifth month and declining thereafter. Polymerase chain reaction for SIV sequences and cultures of peripheral-blood mononuclear cells failed to show infection. Inoculation of an SIV-negative monkey with blood from the worker did not cause infection. Animal-care and laboratory workers should adhere strictly to recommended procedures to avoid accidental exposures when working with SIV-infected animals or specimens.

The objective of this study was to genetically characterize HIV-1 strains in injecting drug users (IDU) in Bangkok Thailand in 1994 and compare these with strains found earlier in Thai IDU; such information is essential for HIV-1 vaccine development and evaluation. Peripheral blood mononuclear cells were collected from 84 IDU attending 14 drug treatment clinics in Bangkok in 1994. DNA was amplified using a nested polymerase chain reaction (PCR) procedure and sequenced directly (without cloning) from the PCR products. The V3 and flanking regions (345 nucleotides) of the env gene were analyzed using a neighbor-joining tree. Only 1 (1%) strain was a typical subtype B virus 69 (82%) were genetically distinct subtype B viruses (Thai B) and 14 (17%) were subtype E strains (Thai A). Persons with recently acquired infection were more likely to have subtype E viruses (p < 0.001) than those in our 1991 survey who were more likely to have subtype B viruses. Pairwise intra-subtype differences within subtypes E and B were 5.3 and 4.3% respectively compared with 3.4 and 3.5% among strains collected in 1991 in Thailand. The genetic diversity within subtypes B and E in Thailand and the proportion of new infections due to subtype E viruses among Bangkok IDU are increasing significantly. These data highlight the importance of monitoring the molecular epidemiology of HIV-1 in populations being considered for HIV-1 vaccine trials. (authors)

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

The explosive spread of HIV-1 strain that infected approximately 75% of injecting drug users (IDUs) in early 1988 in Bangkok clustered phylogenetically with env subtype B viruses typically found in the Americas and Europe, but was genetically distinct from other subtype B strains. This strain was originally named Thai genotype B; hereafter it is referred to as subtype B'. An even more distinct strain, which infected a minority of IDUs but was found in about 90% of persons infected sexually, was referred to as subtype E. To explore the genetic characteristics of the strains of HIV-1 present during the early phase of the Thai epidemic, venous blood was collected in March 1992 from 13 consenting prison inmates. Proviral DNA was extracted from peripheral blood mononuclear cells, amplified using a nested polymerase chain reaction, and 345 nucleotides of the C2-V3 region of the env gene were sequenced. 10 of the 13 prisoners had HIV infections diagnosed in 1986 or 1987. The viral strains from these early HIV infections (THP01-THP10) were distinct from the subtype B' circulating in the general IDU population in Thailand in 1991. Phylogenetic analysis showed that these 10 sequences clustered with other subtype B strains in all 100 replicate trees, but were distinct from the subtype B' viruses. In contrast, the 2 prisoners diagnosed as HIV positive in 1988 and 1992 (THP11 and THP12, respectively) had typical subtype B' sequences containing the unique PLGPGOAW and HLGPGOAW V3 crown motif. The remaining prisoner, THP 13, was infected in 1989 with a subtype E virus. The explanation for the relative lack of spread of the subtype B strains (THP01-THP10) may be that the subtype B' and subtype E strains were introduced by chance into more dynamic, high-risk subgroups that spread the virus more rapidly. The subtype B' and subtype E strains may be also more easily transmitted in the Thai population than the pre-1988 subtype B strains.

Human immunodeficiency virus (HIV) infection, regardless of clinical stage, produces many biologic indicators of virus infection, replication, or both (Fig. 5.1). Such indicators include viremia, antibodies against viral proteins, circulating viral proteins, and nonspecific markers of infection such as neopterin, 32-microglobulins, and changes in the absolute number of and the ratio of CD4 and CD8 cells. Most of the markers can be detected and in many cases semiquantified by serologic tests (Table 5.1). These tests have been used to protect the blood supply, diagnose infections, monitor the progression of disease, monitor the efficacy of drug therapy, and diagnose infections in infants born to HIV-infected mothers. The availability of highly specific, inexpensive tests for HIV antibody, such as the enzyme immunoassay (EIA), have permitted public health agencies to conduct-large scale seroprevalence surveys to define the epidemic.

Children represent an increasingly important part of the human immunodeficiency virus (HIV) epidemic. As of December 1992, there were 4249 HIV-infected children under the age of 13 years who had been reported to the Centers for Disease Control and Prevention (CDC), representing 1.7% of all reported acquired immunodeficiency syndrome (AIDS) cases. During 1992 cases due to perinatal (mother-to-child) transmission and those due to heterosexual contact had the largest increases in number of reported AIDS cases compared with all other transmission groups.

Since the description of the first cases of acquired immunodeficiency syndrome (AIDS) in 1981, the global impact of this new epidemic has been dramatic. By July, 1993, more than 150 countries had reported over 718,894 cases to the World Health Organization (WHO, 1993), and more than 8 million persons worldwide were estimated to be infected with the human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS. There are estimated to be at least 1 million HIV infected persons in the United States alone. Most of these infected persons are expected to develop AIDS. HIV-1 was first isolated in 1983–1984 from patients with ARC and AIDS (Barre-Sinoussi et al., 1983; Gallo et al., 1984; Popovic et al., 1984) and is found worldwide, primarily in Central Africa, Europe, North and South America and now in Asia. A second AIDS virus HIV-2 (Clavel, 1987; Clavel et al., 1986; Horsburgh and Holmberg, 1988), closely related to the simian immunodeficiency virus (SIV) and more distantly related to HIV-1, was discovered in 1986 and shown to be endemic in parts of West Africa and to have limited spread in Western Europe, the United States, Canada, and Brazil.

To investigate the genetic heterogeneity and epidemiological distribution of human immunodeficiency virus type 1 (HIV-1) in Thailand, we determined proviral sequences for 63 HIV-1-infected patients in various risk groups from all over the country between April and July, 1991. Two distinct genotypes of HIV-1, A and B, were found to segregate by mode of transmission. Of 29 sexually infected patients, 25 (86%) had HIV-1 of genotype A and 4 (14%) had genotype B. Among 29 injecting drug users, probably parenterally infected, only 7 (24%) had genotype A and 22 (76%) had genotype B. This segregation is unlikely to have arisen by chance (p0.001). No patient was found to have dual infection. Nucleotide divergence averaged 3.4% among genotype-A-infected patients and 3.5% among genotype-B-infected patients, but 22.0% between the genotypes. 37 of 40 isolates (both genotypes) had the GPGQ tetrapeptide at the tip of the V3 loop, which is common in African HIV-1 strains but rare in North American and European strains, where the GPGR motif predominates. These findings suggest that the waves of HIV-1 infection in injecting drug users and in sexually infected patients in Thailand may not be epidemiologically linked. The nucleotide divergence data point to the separate introductions of the two genotypes in Thailand. Further studies in Thailand and neighbouring countries will be useful in the design and selection of candidate HIV vaccines.

Human immunodeficiency virus type 1 (HIV-1) transmission from infected patients to health-care workers has been well documented, but transmission from an infected health-care worker to a patient has not been reported. After identification of an acquired immunodeficiency syndrome (AIDS) patient who had no known risk factors for HIV infection but who had undergone an invasive procedure performed by a dentist with AIDS, six other patients of this dentist were found to be HIV-infected. Molecular biologic studies were conducted to complement the epidemiologic investigation. Portions of the HIV proviral envelope gene from each of the seven patients, the dentist, and 35 HIV-infected persons from the local geographic area were amplified by polymerase chain reaction and sequenced. Three separate comparative genetic analyses--genetic distance measurements, phylogenetic tree analysis, and amino acid signature pattern analysis--showed that the viruses from the dentist and five dental patients were closely related. These data, together with the epidemiologic investigation, indicated that these patients became infected with HIV while receiving care from a dentist with AIDS.

To determine if patients of a dentist with the acquired immunodeficiency syndrome (AIDS) became infected with human immunodeficiency virus (HIV) during their dental care and, if so, to identify possible mechanisms of transmission.Retrospective epidemiologic follow-up of the dentist, his office practice, and his former patients.The practice of a dentist with AIDS in Florida.A dentist with AIDS, his health care providers and employees, and former patients of the dentist, including eight HIV-infected patients.Identification of risks for HIV transmission (if present), degree of genetic relatedness of the viruses, and identification of infection control and other office practices.Five of the eight HIV-infected patients had no confirmed exposures to HIV other than the dental practice and were infected with HIV strains that were closely related to those of the dentist. Each of the five had invasive dental procedures, done by the dentist after he was diagnosed with AIDS. Four of these five patients shared visit days (P greater than 0.2). Breaches in infection control and other dental office practices to explain these transmissions could not be identified.Although the specific incident that resulted in HIV transmission to these patients remains uncertain, the epidemiologic evidence supports direct dentist-to-patient transmission rather than a patient-to-patient route.

Persons infected with the human immunodeficiency virus (HIV) have a spectrum of clinical features, ranging from persons who are infected but appear completely healthy to those with rapid disease progression and mortality. All groups, regardless of clinical stage, possess several biologic indicators of viral infection or replication (Figure 5.1). These include viremia, antibodies against viral proteins, circulating viral proteins, and nonspecific markers such as neopterin, beta2-microglobulins, and T4 cell counts. A variety of immunologic tests currently exists for the detection of viral antibodies and protein antigens. These assays have permitted testing programs to protect the blood supply from infected units and to conduct seroprevalence surveys to define the epidemic. More recently, the presence or absence of viral and nonspecific markers have been used to predict the onset of clinical disease.

Since the first cases of acquired imunodeficiency syndrome (AIDS) were described in 1981, the global impact of this new epidemic has been dramatic. By December 1990, more than 150 countries had reported over 307,000 cases to the World Health Organization (WHO), and over 8 million persons worldwide were estimated to be infected with the human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS. There are estimated to be at least 1 million HIV-infected persons in the United States alone. Most of these infected persons are expected to develop AIDS. HIV infection throughout the world has continued to be limited to four major modes of transmission: (i) sexual intercourse (vaginal or anal); (ii) transfusion of HIV-contaminated blood or blood products; (iii) sharing of contaminated injection equipment (mainly involved in illegal intravenous drug use); and (iv) transmission from an infected woman to her fetus or infant (perinatal transmission).

Children represent an increasingly important part of the human immunodeficiency virus (HIV) epidemic. As of December 31, 1990, 2,786 children under the age of 13 years had been reported to the Centers for Disease Control (CDC), representing 1.7% of all reported AIDS cases. In the past year, cases due to perinatal (mother-to-child) transmission had the largest increase in number of reported AIDS cases compared with all other transmission groups.1 Prepubertal children acquire HIV primarily through two routes: (i) perinatally through exposure to their infected mothers and (ii) parenterally through transfusions of blood or blood products. Transmission through sexual abuse or through percutaneous exposure to contaminated needles used for illicit drug injection is possible but such are rarely reported. Of the 2,786 children reported, 2,327 (84%) acquired HIV perinatally, 252 (9%) acquired HIV through transfusion of blood or blood products, 139 (5%) were children with coagulation disorders, and 68 (2%) were children with undetermined modes of transmission (usually undetermined because of inadequate information).

The etiologic agent of acquired immunodeficiency syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1), was first isolated during 1983-1984 from patients with AIDS-related complex (ARC) and AIDS.’-3 HIV-1 is the predominant AIDS virus and is found worldwide, primarily in Central Africa, Europe, and North and South America. A second AIDS virus, HIV-2,4-6 closely related to the simian immunodeficiency virus (SIV), was discovered in 1986 and was shown to he endemic in parts of West Africa with limited spread in Western Europe. HIV-2 is only now beginning to appear in the Americas, mainly in the United States, Canada, and Brazil. Infection with either HIV-1 or HIV-2 results in a number of biologic and pathologic changes leading to a spectrum of immune dysfunctions, neurologic disorders, enteropathy, and AIDS.

While Honduras is home to only 15% of Central America's population, it has 60% of the region's AIDS cases. There have been 4973 reported cases of full-blown AIDS in the country and the Health Ministry reports that more than 8000 Hondurans have been infected with HIV since the first Honduran case was diagnosed in 1985. 995 people with AIDS have since died. The authors conducted an investigation to determine which HIV-1 subtype is present in Honduras and the degree of genetic variation among HIV-1 strains by analyzing viral nucleotide sequences from the envelope region of HIV-1 isolates obtained from the two most affected regions of the country. They determined the predominant HIV-1 subtype among 27 HIV-1-infected patients attending sexually transmitted disease clinics in San Pedro Sula and Tegucigalpa by sequencing and analyzing the C2V3 regions of the envelope glycoprotein gp 120. Genomic DNA was isolated from patients' peripheral blood mononuclear cells. Phylogenetic analysis determined that all 27 Honduran sequences clustered with known subtype B sequences.

Since human immunodeficiency virus (HIV) transmission from a dentist to six of his patients was first reported in 1990 by the Florida Department of Health and Rehabilitative Services and the Centers for Disease Control and Prevention, controversy and speculation have surrounded the investigation of that case. This controversy has been fueled by the inability to determine exactly how the transmissions occurred. Many theories have appeared in the media and have led to confusion and uncertainty about the facts of this investigation. Recently, a magazine article and a newspaper article, as well as a segment on the television newsmagazine "60 Minutes," presented information that was largely based on findings by investigators hired as part of private litigation and that cast doubt on the conclusion that the patients had been infected by the dentist. However, these reports omitted pertinent epidemiologic and laboratory evidence that shows that no other sources of HIV infection could be documented for the six dental patients. The scientific evidence indicates that the Florida dentist transmitted HIV to six of his patients.

The polymerase chain reaction (PCR) and reverse transcription were used to assess human immunodeficiency virus type 1 (HIV1) RNA expression in peripheral blood mononuclear cell samples from seropositive subjects. HIV RNA was detected from seropositive subjects who had no symptoms, lymphadenopathy syndrome, and acquired immunodeficiency syndrome. DNA PCR of the samples used for RNA extraction showed that seventeen of eighteen (94%) contained HIV proviral DNA. Eleven (65%) of the seventeen DNA-positive samples were also positive for HIV RNA, including samples from four patients undergoing antiviral drug treatment. Serum HIV antigen assays detected only six (32%) of the nineteen PCR-positive samples. Owing to the speed and high sensitivity of PCR for HIV detection, this technique will be suitable for monitoring antiviral therapy and the virus load of people with HIV infections.

Polymerase chain reaction (PCR) was used to detect human immunodeficiency virus (HIV)-1 DNA in peripheral blood mononuclear cells to assess in hemophilic men whether any were HIV-seropositive but uninfected or seronegative but infected and in seronegative sex partners of seropositive hemophilic men whether any were infected. Of 40 seropositive men, 38 (95%) were PCR-positive; one was PCR-indeterminate and one PCR-negative. None of 41 seronegative men who used only donor-screened, virus-inactivated coagulation factor products were PCR-positive. However, two of six who received noninactivated products were PCR-positive; one had low T-helper cell counts and died of unrelated causes and the other had seroconverted 11 mo later. PCR with a second primer pair also detected HIV-1 DNA in these two men. None of 25 seronegative female sex partners of seropositive men, including six men with AIDS and seven with AIDS-related symptoms, were PCR-positive. These data suggest that most seropositive hemophilic men are HIV-infected; whether some are infected with defective virus remains to be resolved as does the infection status of seropositive PCR-negative men. Identification of two seronegative PCR-positive men supports the possibility that HIV-1 DNA can be detected before seroconversion.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.

The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells.

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on 51 Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing 125 I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.

The results of a seroprevalence study of infection with human immunodeficiency virus (HIV)-1 and 2 in selected populations in Abidjan (Ivory Coast) suggest that HIV-1 infection is more widespread in west Africa than is generally acknowledged. The 956 study subjects who were tested by enzyme-linked immunosorbent assay in September 1987 included 200 consecutive hospitalized patients on infectious diseases and internal medicine wards at Abidjans largest hospital 200 consecutive outpatients at Abidjans 2 largest tuberculosis treatment centers 200 volunteer blood donors 200 consecutive women from an urban prenatal clinic and 156 consecutive outpatients attending urban sexually transmitted disease (STD) clinics. The prevalence of HIV-1 infection (in ascending order) was 6% among STD patients 7% among pregnant women 8% among blood donors 15% among internal medicine inpatients 16% among tuberculosis patients and 24% among infectious diseases inpatients. The prevalence of infection with HIV-2 was 1% among STD patients 2% among both pregnant women and internal medicine inpatients 4% among both blood donors and tuberculosis patients and 6% among infectious disease inpatients. Dual infection with both viruses was found in 1% of pregnant women 2% of STD patients 3% of donors 6% of internal medicine inpatients 8% of tuberculosis patients and 15% of infectious disease patients. 25% of all seropositive individuals reacted serologically to both viruses. Of the 29 subjects who met the WHO clinical criteria for acquired immunodeficiency syndrome in the infectious diseases service 13 were seropositive for HIV-1 4 for HIV-2 and 4 were dually reactive.

Article de synthese: Virus HIV: structure, immunogenicite, methodes de detection chez l'adulte et chez le nourrisson. Virus HTLV: structure et fonction, methodes de diagnostic

To estimate the duration and frequency of the period of HIV infection without detectable antibody, modelling techniques were applied to results of detection of HIV DNA by means of the polymerase chain reaction (PCR) and to data from cases in published reports. PCR was carried out with gag and env region primers on samples from 27 homosexual and 12 haemophilic men for whom stored samples were available from before and after seroconversion; serum was also tested for p24 antigen by antigen-capture enzyme immunoassay. HIV DNA was detectable before seroconversion in 4 men; in all 4 PCR was positive only in the seronegative sample taken closest to the time of seroconversion. In 3 men antigen was detected before seroconversion; in each case HIV DNA was also detected. By a Markov model, the time from infection with HIV (as assessed by detection of HIV DNA) to first detection of HIV antibody was estimated to be 2.4 (SE 2.1) months for the median individual. Modelling of cases of HIV infection with known exposure in published reports gave a median estimate of 2.1 (0.1) months from exposure to antibody detection, and 95% of cases would be expected to seroconvert within 5.8 (0.6) months. HIV infection for longer than 6 months without detectable antibody seems uncommon.

Les sujets avec des allogreffes recevant des medicaments immunodepresseurs forment un anticorps detectable contre le HIV dans la periode prevue. Lorsqu'un dosage immunoenzymatique recombinant et un dosage des anticorps deviennent possibles ces dosages peuvent etre utilises en association pour evaluer les sujets a une exposition recente au HIV