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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.

In comparison to current on-market assays, the ARCHITECT rHTLV-I/II assay is the first fully automated assay that simultaneously detects human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) in human serum and plasma. Specificity was assessed on 5646 blood donors and 692 clinical specimens. For sensitivity determination, 301 HTLV-I-positive and 105 HTLV-II-positive specimens were tested. Precision was between 3.98% and 4.31% coefficient of variation (CV) for specimens with 1 to 6 sample to cutoff. Specificity was 99.95% and 99.86% on specimens from blood donors and hospitalized patients, respectively. Sensitivity evaluation showed 100% detection on 301 HTLV-I and 105 HTLV-II specimens. HTLV-I and HTLV-II viruses are still circulating among general populations even in the low prevalence areas. To control the further spread of these retroviruses, we need to know that it is important to continue screening of blood. The performance evaluation data from this study demonstrate that the high throughput and fully automated ARCHITECT rHTLV-I/II chemiluminescence immunoassay effectively serves this purpose.

Objective: The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. Methods: From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaounde, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. Results: HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRF02_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. Conclusions: HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

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.

An HIV-1 group N infection, 02CM-DJO0135, was identified among specimens collected in 2002 at the D'Joungolo Hospital, Yaoundé, Cameroon. Sequences were obtained from viral RNA extracted from plasma for regions of LTR-gag, pol-vif, and env. The virus amplified from the specimen is closely related to a previously reported group N virus, 02CM-DJO0131, that was also collected at this hospital in 2002. Although the viral sequences for the two isolates differ, their close relationship suggests that the two specimens are linked. No patient histories are available for 02CM-DJO0131 and 02CM-DJO0135; the specimens could have been drawn from a husband/wife, mother/child, or a single individual. However, differences in seroreactivity indicate that it is unlikely that the specimens were drawn from the same patient. This report documents the second case that suggests linkage between group N-infected individuals and indicates that there is ongoing transmission of HIV-1 group N in Cameroon.

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.

In this study, we evaluated the performance of six HIV combined p24 antigen and antibody (Ag/Ab) assays versus two third-generation anti-HIV antibody assays. The assays were evaluated using p24 antigen panel of 31 HIV-1 subtypes (n = 124), 25 HIV-1 seroconversion panels (n = 176), HIV-1 antibody positive samples including group M subtypes and group O (n = 559), HIV-2 positive samples (n = 110), and unselected HIV negative samples from four French private laboratories (n = 1005). The results showed that overall HIV combined Ag/Ab assays present better performance, when compared to antibody-only assays. However, some differences were observed in the sensitivity of the six HIV combined Ag/Ab assays evaluated. The AxSYM and Murex Combo assays had the best sensitivity score in this study and reduced the window period by 2.0-2.35 days relative to antibody only assays and 1-2.17 days relative to the other combined Ag/Ab assays. Among combined HIV Ag/Ab assays, Genscreen Plus and AxSYM Combo presented the highest specificity, with 99.9% and 99.8%, respectively.

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 .

V3 serotyping refers to a system based on binding of antibody in patient sera to V3-loop peptides derived from HIV-1 env genetic subtypes. The V3X serotype represents reactivity of serum from an HIV-1-infected patient (regardless of viral genetic subtype), which reacts preferentially to a V3 peptide derived from the X subtype sequence. We have classified HIV-1 serotypes, determined the relationship between the HIV-1 V3 serotypes and viral genetic subtypes in a large study (n = 125), and evaluated the performance of three different V3 peptide-binding assays. Seven HIV-1 V3 serotypes were identified: A, B, B-Br, B-Th, C, D, and E. Serotypes B-Br and B-Th represent sera that react specifically to peptides derived from Brazilian B (B-Br, GWGR) and Thai B (B-Th, GPGQ) strains. The HIV-1 V3 B, C, and E serotypes correlated closely with their viral env genetic subtypes; 19–26 of 32 B sera (59–79%), 3–4 of 4 C sera (75–100%), and 19–22 of 23 E sera (83–96%) were identified as serotypes B, C, and E, respe...

Extensive transmission of human immunodeficiency virus type 1 (HIV-1) in Thailand began in 1988, resulting in an estimated 800,000 cumulative infections by 1994. During 1994 and 1995, we collected blood specimens from 215 asymptomatic HIV-1-infected people with various risk behaviors from nine locations in all four regions of Thailand. HIV-1 subtypes and genetic heterogeneity were determined for 214 strains by a combination of direct DNA sequencing (n = 95), subtype-specific oligonucleotide probe testing (n = 201), and V3-loop peptide enzyme immunoassay (PEIA) (n = 214). All strains were either env subtype E (175; 81.8%) or B (39; 18.2%). Of the subtype B isolates, 37 (94.9%) were B' and 2 (5.1%) were more typical North American-like B strains (most subtype B strains in Thailand are part of a distinct subcluster within the subtype B branch on phylogenetic trees, termed B'; formerly Thai B or BB). Of 149 viruses from people with sexual risk behaviors from all regions, 146 (98.0%) were subtype E. Of 65 viruses from injecting drug users (IDUs), 29 (44.6%) were subtype E and 36 (55.4%) were subtype B, including 35 B' strains. There was regional variation in the proportions of subtypes E and B' among IDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene (mid-C2 to the start of the V4 region) was low (5.7% for subtype E and 3.1% for subtype B') compared with other HIV-1 group M subtypes from different countries. These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable setting for evaluating candidate HIV-1 vaccines. The mix of subtype E and B' strains among IDUs also offers the opportunity to study phenotypic differences between the two subtypes.The extensive transmission of HIV-1 in Thailand which began in 1988 led to an estimated 800,000 cumulative infections in the country by 1994. The authors collected blood specimens during 1994 and 1995 from 215 asymptomatic HIV-1-infected people with various risk behaviors from 9 locations across Thailand. HIV-1 subtypes and genetic heterogeneity were then determined for 214 strains using a combination of direct DNA sequencing, subtype-specific oligonucleotide probe testing, and V3-loop peptide enzyme immunoassay. 175 strains were subtype E and 39 were subtype B. 37 of the subtype B isolates were B' and 2 were more typical North American-like B strains. Of 149 viruses from people with sexual risk behaviors from all regions of the country, 146 were subtype E. Of 65 viruses from IV drug users (IVDUs), 29 were subtype E and 36 were subtype B, including 35 subtype B' strains. Regional variation was observed in the proportions of subtypes E and B' among IVDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene was 5.7% for subtype E and 3.1% for subtype B'. The finding of 2 HIV-1 subtypes with low heterogeneity suggests that Thailand may be an appropriate setting in which to evaluate candidate HIV-1 vaccines. The mix of subtype E and B' strains among IVDUs will also allow the study of phenotypic differences between the 2 subtypes.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.

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.

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at ∼25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was ∼125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.

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.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.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.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.

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.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.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.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 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.

Although XMRV dissemination in humans is a matter of debate, the prostate of select patients seem to harbor XMRV, which raises questions about its potential route of transmission. We established a model of infection in rhesus macaques inoculated with XMRV. In spite of the intravenous inoculation, all infected macaques exhibited readily detectable XMRV signal in the reproductive tract of all 4 males and 1 female during both acute and chronic infection stages. XMRV showed explosive growth in the acini of prostate during acute but not chronic infection. In seminal vesicles, epididymis, and testes, XMRV protein production was detected throughout infection in interstitial or epithelial cells. In the female monkey, epithelial cells in the cervix and vagina were also positive for XMRV gag. The ready detection of XMRV in the reproductive tract of male and female macaques infected intravenously suggests the potential for sexual transmission for XMRV.

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.

We have developed a combination immunoblot (combi-blot) for simultaneous detection and differentiation of HIV-1 and HIV-2 antibodies using HIV-1 lysate and HIV-1 and HIV-2 synthetic peptide antigens in a single assay. Minimal modification in antigen strip preparation and no modification in assay procedure of the current HIV-1 Western blot protocol was required. Implementation of this combi-blot following the use of the combination HIV-1/-2 enzyme immunoassay would simplify the current HIV testing algorithm and increase the accuracy of HIV-2 surveillance.

This study sought to examine the genetic heterogeneity of the V3 region of HIV-1 gp 120 from 22 Brazilian HIV-1 specimens. Genetic heterogeneity was examined by DNA sequencing of the C2 V3 region of the HIV-1 envelope (env) gene from polymerase chain reaction (PCR)-amplified HIV-1 DNA. Deduced amino-acid sequences were compared to determine the extent of amino-acid conservation among the Brazilian specimens. Genetic similarity among and between the Brazilian specimens and other previously published HIV-1 isolates was analyzed by principal coordinate and DNA parsimony methods. A 282 base pair (bp) region of a 1.5 kilo (k) bp PCR-amplified HIV-1 env fragment was sequenced by a Tag dye-labeled primer cycle sequencing reaction. Nucleotide sequences were used to analyze interspecimen relationships based on overall nucleotide sequence similarity and DNA parsimony principles. Amino-acid comparison showed that 15 of the 35 (43%) residues of the V3 loop were conserved among the Brazilian specimens. 9 of the 22 (40% Brazilian specimens contained the North American-European GPGR tetrapeptide motif while 8 (36%) contained the GWGR motif previously reported in Japanese isolates. Principal coordinate analysis demonstrated that 19 of 20 examined Brazilian HIV-1 specimens were more similar to North American and Haitian isolates than to African isolates. Similar results were also obtained by DNA parsimony analysis. The majority of the Brazilian specimens examined are more genetically related to North American and Haitian HIV-1 isolates than to African isolates. This finding an the presence of a GWGR V3 loop motif in some Brazilian isolates may be important for vaccine development. (authors)

The envelope (env) gene of human immunodeficiency virus type 1 (HIV-1) was amplified by polymerase chain reaction (PCR) from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1-infected women from Kinshasa, Zaire. Amplified DNA was directly sequenced with a primer specific for the HIV-1 env C2 region. The predicted amino acid sequences for the C2-V3 region for the 14 specimens are presented. The tetrapeptide sequence, GPGQ, located at the crown of the V3 loop, is conserved in all specimens. The same tetrapeptide sequence is present in the Zairian isolate MAL, but not in other published Zairian isolates (Z6, ELI, Z321, JY1, and NDK). Sequence comparison of the env C2-V3 region among the 14 specimens from Kinshasa revealed a 9-25% range of nucleotide divergence, with an average of 16%. Divergence between the 14 specimens and the Zairian isolates MAL, Z6, ELI, Z321, JY1, and NDK ranged from 13 to 31%. A range of 18-28% nucleotide sequence divergence was demonstrated between the 14 Kinshasa specimens and the North American isolate MN. These results demonstrate the importance of examining HIV-1 samples from diverse geographic origins in the development of effective HIV-1 vaccines.

A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.

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.

Background Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined. Results Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively. Conclusions This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.

Analysis of sera from hospitalized Brazilian patients by whole-virus lysate-based enzyme immunoassay and western blot indicated that 0.4% were reactive to HIV-2 alone while 4% were reactive to both HIV-1 and HIV-2. When these sera were tested for HIV antibody by type-specific peptide enzyme immunoassays dual seropositivity was confirmed in only 0.4% of patients. To define genetically the HIV strains within the population the authors analyzed peripheral blood mononuclear cells from selected seropositive patients for the presence of HIV-1 and HIV-2 proviral DNA using the polymerase chain reaction (PCR). Independent primers/probes/sets were used for the amplification and detection of viral sequences from the longterm terminal repeat gag and protease gene regions. Author findings confirmed the serologic evidence of HIV-2 in Brazil and determined the extend of mixed HIV-1 and HIV-2 infections. Detailed evaluation of the amplified viral protease sequences by endonuclease restriction analysis and DNA sequencing independently confirmed mixed HIV-1 and HIV-2 infections in the 2 patients who were seropositive for HIV-1 and HIV-2. The data further indicated that these isolates are distinct from the HIV laboratory standards. The authors interpret the combination of culture and PCR findings to demonstrate the presence of both HIV-1 and HIV-2 in Brazil. (authors)

Human immunodeficiency virus (HIV types 1 and 2) replication is controlled by the interaction of viral-encoded regulatory proteins and host cellular proteins with the viral long terminal repeat (LTR). The presence of HIV-1 and HIV-2 trans-activator proteins, tat1 and tat2, respectively, greatly increases viral gene expression from their homologous LTRs. It is unclear if the cellular factors that support tat1-directed trans-activation of the HIV-1 LTR are the same for tat2 trans-activation of the HIV-2 LTR. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV-1 and HIV-2 tat-directed transactivation. DNA transfection experiments showed that the presence of human chromosome 12 in human-hamster hybrid clones was necessary for high-level tat-directed trans-activation of the HIV-1 and -2 LTR. Cross-trans-activation of the HIV-2 LTR by tat1 was found to be chromosome 12 independent. In addition, chromosome 12 did not support trans-activation of another human retrovirus (human T-cell leukemia virus type I). Our results suggest that HIV-1 and -2 have evolved to employ a cellular pathway(s) encoded on human chromosome 12 for supporting homologous tat-directed trans-activation. Trans-activation of the HIV-2 LTR by tat1 in chromosome 12-minus cells suggests that multiple cellular pathways can be recruited to trans-activate the HIV-2 LTR and that these pathways may have been important in an HIV-like progenitor virus.

Recent reports have suggested that maternal antibodies to specific epitopes of the variable region 3 (V3 loop) of gp120 of HIV-1 might protect against perinatal transmission. In an attempt to confirm these findings, sera from 34 HIV-1-seropositive mothers, representing 13 episodes of mother-to-infant transmission and 23 episodes of non-transmission (two mothers had two pregnancies each during the study period), were tested for the presence of antibodies to various regions of the gp120 V3 loop. Synthetic peptides were generated from HIV-1MN. Of the four peptides tested by enzyme-linked immunosorbent assay (ELISA), only antibody to the C53 peptide (Env310-322, principal neutralizing determinant) was present in maternal sera. Antibody to the C53 sequence was present in 11 specimens from transmitting mothers and 21 from non-transmitting mothers (84.6 and 91.3%, respectively, P = 0.6). No reactivity was detected against the C51, C57, or C58 peptide sequences, located on the sides of the V3 loop. In an antigen-limited ELISA, only two specimens from transmitting mothers and two specimens from non-transmitting mothers had detectable 'high-affinity' antibodies to C53 at low antigen concentrations (15.4 and 8.7%, respectively; P = 0.6). Our results do not support previous reports that epitope-specific antibodies to the V3 loop peptides protect against perinatal transmission. Further research is required to determine whether any specific maternal humoral response might influence HIV-1 perinatal transmission.