Resources for genetic management and genomics research on non-human primates at the National Primate Research Centers (NPRCs).

The National Primate Research Centers (NPRCs) established Working Groups (WGs) for developing resources and mechanisms to facilitate collaborations among non-human primate (NHP) researchers. Here we report the progress of the Genome Banking and the Genetics and Genomics WGs in developing resources to advance the exchange, analysis and comparison of NHP genetic and genomic data across the NPRCs. The Genome Banking WG has established a National NHP DNA bank comprising 1250 DNA samples from unrelated animals and family trios from the 10 NHP species housed within the NPRC system. The Genetics and Genomics WG is developing SNP arrays that will provide a uniform, highly informative, efficient and low-cost method for rhesus and long-tailed macaque genotyping across the eight NPRCs. This WG is also establishing a Biomedical Informatics Research Network-based portal for shared bioinformatics resources including vital statistics, genotype and population data and information on the National NHP DNA bank.

New multiple antigenic peptide-based enzyme immunoassay for detection of simian immunodeficiency virus infection in nonhuman primates and humans.

Infections with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) are zoonotic infections. In Africa, the potential exists for additional cross-species transmissions from at least 33 different species of simian immunodeficiency virus (SIV)-infected nonhuman primates (NHPs) through hunting and butchering of these animals for food. Here we describe a highly sensitive and specific enzyme immunoassay (EIA) with chemically modified, multiple antigenic peptides (MAPs) developed for the detection and discrimination of antibodies to SIV genetic lineages. The SIV EIA was developed by using a comprehensive array of MAPs covering two envelope gene regions from all of the SIV lineages for which env sequences were available. Assay sensitivity was evaluated by using 63 plasma or serum samples obtained from primates naturally or experimentally infected with SIVs from 10 genetic lineages. Assay specificity was determined by using 97 known SIV-negative plasma specimens from these same species. Also used in the evaluations were 369 human samples: 198 HIV seronegative, 170 HIV-1 and/or HIV-2 seropositive, and 1 from a human SIVsm infection. Overall assay sensitivity and specificity were 100% with both immunodominant region (IDR) and V3 region MAPs. Although SIV env sequences from talapoin monkeys were not available for specific MAP inclusion, 5 (100%) of 5 SIVtal-infected samples were detected through cross-reactivity with other SIV IDR MAPs used in the assay. The one human SIVsm infection was identified. In conclusion, our SIV MAP EIA proved to be highly sensitive and specific for detecting SIV infections in NHPs and humans. As shown with SIV-infected talapoin monkeys, this assay has the potential to detect previously unidentified SIV strains and should be suitable for sentinel surveillance for potential new cross-species transmissions of SIVs to humans.

Human infection with foamy viruses.

Virtually all nonhuman primate species investigated thus far including prosimians, New World and Old World monkeys and apes all harbor distinct and species-specific clades of simian foamy virus (SFV). However, evidence supporting the existence of a human-specific foamy virus (FV) is not yet available. Early reports describing widespread infection of healthy and sick humans with FV could not be confirmed. In contrast, all FV infections documented in humans are of zoonotic origin and are identified in persons occupationally exposed to nonhuman primates. The introduction of SFV into humans raises several public health questions regarding disease outcomes and potential for human-to-human transmissibility. The available data from a very limited number of SFV-infected humans suggest that these infections are nonpathogenic and are not easily transmissible. Additional studies are needed to better define the prevalence and natural history of SFV in humans.

Simian foamy virus infection in a blood donor.

BACKGROUND: Infections with simian foamy virus (SFV) are widely prevalent in nonhuman primates. SFV infection was confirmed in a worker, occupationally exposed to nonhuman primates, who donated blood after the retrospectively documented date of infection. Human-to-human transmission of SFV through transfusion and its pathogenicity have not been studied. STUDY DESIGN AND METHODS: Recipients of blood from this donor were identified and blood samples from such recipients were tested for SFV infection by Western blot and PCR assay. RESULTS: One recipient of RBCs and another recipient of FFP had died; retroviral infections were not implicated. One platelet recipient could not be tested. Recipients of RBCs (two), a WBC-reduced RBC unit (one), and a platelet unit (one) tested SFV-negative 19 months to 7 years after transfusion. Tested recipients had transfusions 3 to 35 days after blood donation. Samples of one lot of albumin and three lots of plasma protein fraction (manufactured from recovered plasma from two donations) tested negative both for antibodies and for viral RNA. CONCLUSION: SFV transmission through transfusion was not identified among four recipients of cellular blood components from one SFV-infected donor. Derivatives containing plasma from that donor tested negative for SFV.

Contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection.

The life cycle of human immunodeficiency virus type 1 (HIV-1) is intricately related to the activation state of the host cells supporting viral replication. Although cellular activation is essential to mount an effective host immune response to invading pathogens, paradoxically the marked systemic immune activation that accompanies HIV-1 infection in vivo may play an important role in sustaining phenomenal rates of HIV-1 replication in infected persons. Moreover, by inducing CD4+ cell loss by apoptosis, immune activation may further be central to the increased rate of CD4+ cell turnover and eventual development of CD4+ lymphocytopenia. In addition to HIV-1-induced immune activation, exogenous immune stimuli such as opportunistic infections may further impact the rate of HIV-1 replication systemically or at localized anatomical sites. Such stimuli may also lead to genotypic and phenotypic changes in the virus pool. Together, these various immunological effects on the biology of HIV-1 may potentially enhance disease progression in HIV-infected persons and may ultimately outweigh the beneficial aspects of antiviral immune responses. This may be particularly important for those living in developing countries, where there is little or no access to antiretroviral drugs and where frequent exposure to pathogenic organisms sustains a chronically heightened state of immune activation. Moreover, immune activation associated with sexually transmitted diseases, chorioamnionitis, and mastitis may have important local effects on HIV-1 replication that may increase the risk of sexual or mother-to-child transmission of HIV-1. The aim of this paper is to provide a broad review of the interrelationship between immune activation and the immunopathogenesis, transmission, progression, and treatment of HIV-1 infection in vivo.

Production of a novel viral suppressive activity associated with resistance to infection among female sex workers exposed to HIV type 1.

To investigate mechanisms of natural resistance to human immunodeficiency virus type 1 (HIV-1), we obtained blood samples from eight women who remained HIV-1 negative after > 3 years of high-risk sex work in Chiang Rai, Thailand. CD4+ T lymphocytes from these highly exposed, persistently seronegative (HEPS) women were readily infectable in vitro with HIV-1 subtypes B and E. Autologous CD8+ cell suppression of both HIV-1 subtypes was evident in HEPS infection cultures, but to an extent also observed in cultures from non-HIV-exposed individuals. Furthermore, production of beta-chemokines was not enhanced in HEPS cultures. However, HEPS cultures displayed significantly enhanced production of a soluble activity that suppressed postintegrated HIV-1 replication. This activity was the unique product of CD4+ T cell and monocyte cocultures. Therefore, although HEPS individuals are apparently susceptible to infection, the production of a postintegrated HIV-1 suppressive activity during monocyte-T cell interactions might protect against the establishment of infection by limiting viral dissemination.

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping.

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.

Predominance of HIV type 1 subtype G among commercial sex workers from Kinshasa, Democratic Republic of Congo.

We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early part of the HIV-1 epidemic among commercial sex workers (CSWs) in Kinshasa, Democratic Republic of Congo (formerly Zaire). Serologic analysis revealed that 27 (28.7%) of the 94 specimens were seropositive by both peptide and whole-virus lysate EIAs and that 24 were positive by molecular screening assays, using generic primers that can detect all known groups of HIV-1. Phylogenetic analyses of the gag(p24), C2V3, and gp41 regions of these 24 specimens showed that all were group M; none of them had any evidence of group O, N, or SIVcpz-like sequences. On the basis of env sequence analysis, the 24 group M specimens were classified as subtypes G (37.5%), A (21%), F1 (12.5%), CRF01_AE (8%), D (4%), and H (4%); 3 (12.5%) were unclassifiable (U). Similar analysis of the gag(p24) region revealed that the majority of infections were subtype A; however, one-third of the specimens were subtype G. Parallel analysis of gag(p24) and env regions revealed discordant subtypes in many specimens that may reflect possible dual and/or recombinant viruses. These data suggest a predominance of subtype G (both pure G and recombinant CRF02_AG) during the early part of the epidemic in Kinshasa. Infections with group N or SIVcpz-like viruses were not present among these CSWs in Kinshasa.

Pig-tailed macaques infected with human immunodeficiency virus (HIV) type 2GB122 or simian/HIV89.6p express virus in semen during primary infection: new model for genital tract shedding and transmission.

Characterizing human immunodeficiency virus (HIV) expression in semen during primary infection remains essential to understanding the risk of sexual transmission. This investigation represents the first systematic evaluation of male genital tract shedding to use a nonhuman primate model, including the impact of exposure route and viral virulence. Male macaques were inoculated with either a chronic disease-causing virus (HIV-2(GB122); n=4 intravenous; n=4 intrarectal) or an acutely pathogenic simian/HIV strain (SHIV(89.6P); n=2 intravenous). All macaques were systemically infected, and seminal plasma virion-associated RNA (vRNA) levels were approximately 10-fold lower than those in blood. In HIV-2(GB122) infection, seminal virus was delayed by 1-2 weeks compared with that in blood. Intrarectal inoculation resulted in a shorter duration of seminal vRNA expression and intermittent seminal cell provirus. No delays, higher peaks ( approximately 50-fold), or longer durations in seminal virus expression were noted for SHIV(89.6P) infection. This novel model definitively establishes that virus dissemination results in early peak seminal levels and provides a basis for evaluating interventions targeting male genital tract expression.

Lack of evidence of endogenous avian leukosis virus and endogenous avian retrovirus transmission to measles, mumps, and rubella vaccine recipients.

The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella (MMR) vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and validated. All serum samples were negative for antibodies to endogenous ALV by Western blot analysis. Peripheral blood lymphocyte samples from 100 vaccinees were further tested by polymerase chain reaction for both ALV and EAV proviral sequences; all were negative. Matching serum samples were tested by reverse transcriptase polymerase chain reaction for ALV and EAV RNA, and all 100 samples were negative, providing no evidence of viremia. These findings do not indicate the presence of either ALV or EAV infection in MMR vaccine recipients and provide support for current immunization policies.

Early detection of reverse transcriptase activity in plasma of neonates infected with HIV-1: a comparative analysis with RNA-based and DNA-based testing using polymerase chain reaction.

Plasma viral load from 71 HIV-1-infected neonates was measured by using Amp-RT, an ultrasensitive quantitative reverse transcriptase (RT) assay and by nucleic acid sequence-based amplification (NASBA), an RNA-based quantitative assay. Results were then compared with those obtained from detection of proviral DNA in peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction (PCR) using Turnbull analysis. At 5 days of life, 50% of neonates were positive by Amp-RT, 30% were NASBA positive, and 20% were DNA-PCR positive. Through the first 12 days of life, Amp-RT was more sensitive than either NASBA or DNA-PCR in detecting HIV-1 infection. Amp-RT values correlated well with NASBA RNA values, with an overall Pearson's r = 0.63 (95% confidence interval [CI], 0.40-0.78). In proportional hazards analysis of infants aged 14 to 61 days (N = 31), a one-log increase in RNA-based viral load was associated with a > fivefold risk of disease progression when using the U.S. Centers for Disease Control and Prevention (CDC) clinical Category C (CDC-C) or death as an endpoint (p =.014). Kaplan-Meier analysis of these data found that RNA viral loads were able to predict disease progression using CDC-C/death as an endpoint (p = .013). Early quantitative viral load measurements may assist clinicians in diagnosing HIV-1 infection, stratifying risk of disease progression, and implementing a treatment plan using highly active antiretroviral therapy for infants within the first few weeks of life.

Evidence of infection with simian type D retrovirus in persons occupationally exposed to nonhuman primates.

Simian type D retrovirus (SRV) is enzootic in many populations of Asian monkeys of the genus Macaca and is associated with immunodeficiency diseases. However, the zoonotic potential of this agent has not been well defined. Screening for antibodies to SRV was performed as part of an ongoing study looking for evidence of infection with simian retroviruses among persons occupationally exposed to nonhuman primates (NHPs). Of 231 persons tested, 2 (0.9%) were found to be strongly seropositive, showing reactivity against multiple SRV antigens representing gag, pol, and env gene products by Western immunoblotting. Persistent long-standing seropositivity, as well as neutralizing antibody specific to SRV type 2, was documented in one individual (subject 1), while waning antibody with eventual seroreversion was observed in a second (subject 2). Repeated attempts to detect SRV by isolation in tissue culture and by using sensitive PCR assays for amplification of two SRV gene regions (gag and pol) were negative. Both individuals remain apparently healthy. We were also unable to transmit this seropositivity to an SRV-negative macaque by using inoculation of whole blood from subject 1. The results of this study provide evidence that occupational exposure to NHPs may increase the risk of infection with SRV and underscore the importance of both occupational safety practices and efforts to eliminate this virus from established macaque colonies.

Evidence of porcine endogenous retroviruses in porcine factor VIII and evaluation of transmission to recipients with hemophilia.

Since 1984, unheated porcine clotting factor VIII (Hyate:C) has been used to treat severe bleeding episodes in persons with hemophilia who have antibodies to human clotting factor. We document the presence of porcine endogenous retrovirus (PERV) in plasma samples of pigs and in clinical lots of Hyate:C. Both gag and pol PERV RNA sequences were detected by reverse-transcriptase (RT) polymerase chain reaction in 13 of 13 lots of Hyate:C tested. Among 10 of these lots, RT activity also was detected, which confirms the presence of retroviral particles. To assess the transmission of PERV to Hyate:C recipients, we tested serum specimens from 88 recipients of Hyate:C and 23 noninfused control subjects for anti-PERV antibodies by using a Western blot assay. None of the samples was positive. Our data document that PERV particles are a common contaminant of Hyate:C products and suggest that the risk of PERV transmission from these percutaneous exposures is very low.

Infectious disease issues in xenotransplantation.

Xenotransplantation, the transplantation of living organs, tissues, or cells from one species to another, is viewed as a potential solution to the existing shortage of human organs for transplantation. While whole-organ xenotransplantation is still in the preclinical stage, cellular xenotransplantation and extracorporeal perfusion applications are showing promise in early clinical trials. Advances in immunosuppressive therapy, gene engineering, and cloning of animals bring a broader array of xenotransplantation protocols closer to clinical trials. Despite several potential advantages over allotransplantation, xenotransplantation encompasses a number of problems. Immunologic rejection remains the primary hindrance. The potential to introduce infections across species barriers, another major concern, is the main focus of this review. Nonhuman primates are unlikely to be a main source for xenotransplantation products despite their phylogenetic proximity to humans. Genetically engineered pigs, bred under special conditions, are currently envisaged as the major source. Thus far, there has been no evidence for human infections caused by pig xenotransplantation products. However, the existence of xenotropic endogenous retroviruses and the clinical evidence of long-lasting porcine cell microchimerism indicate the potential for xenogeneic infections. Thus, further trials should continue under regulatory oversight, with close clinical and laboratory monitoring for potential xenogeneic infections.

Facilitated detection of adventitious agents using genetically engineered cell lines.

Proposals to expand the number and types of cellular substrates used in the production of live attenuated vaccines, especially to include those of tumour origin, have raised concerns about the current capacity to detect adventitious agents that may be present in vaccine stocks. Detection of unknown agents is especially difficult because the culture systems used may not be optimal. We hypothesize that failure to grow certain viruses in culture may be the result of the cellular suicide mechanism, known as apoptosis, killing virus-infected cells before the virus can effectively replicate and spread. Our earlier work with an HIV-1 culture system which overexpresses the cellular anti-apoptotic gene, bcl-2, demonstrated that interfering with the apoptotic programme could facilitate HIV-1 expression and accelerate the kinetics of an acute spreading HIV-1 infection. These findings may have implications for improving cell culture detection systems to screen for potentially harmful infectious agents in current and developmental vaccine substrates. In this paper, we briefly review earlier work and discuss future studies aimed at manipulating the cellular apoptotic programme to facilitate the replication of adventitious and transforming viral agents in vitro.

Chronic immune stimulation accelerates SIV-induced disease progression.

The contribution of chronic immune stimulation on the progression of lentivirus-induced disease was evaluated in the SIVmac251 macaque model of AIDS. Following SIV inoculation, seroconversion and control of the acute viral replication phase, repeated immune stimulations with tetanus toxoid (TT), keyhole limpet hemocyanin (KLH) and allogeneic peripheral blood mononuclear cells (PBMC) were initiated in four monkeys. These animals showed a significant shortening of survival when compared with eight non-immune-stimulated control animals inoculated with the same route, dose and stock of SIVmac251 (median survival 9.5 months versus 17 months, P = 0.010). In addition, when the comparison was extended to another 22 control animals of different origin but inoculated by the same route with similar doses and stocks of SIVmac251, the difference in survival was still significant (9.5 versus 18 months, P = 0.003). This accelerated progression of symptomatic disease was not accompanied with significant increases in plasma viral loads, but suboptimal antibody responses to the immunizing antigens were noted, correlating with the length of survival. These findings may have implications for HIV-infected humans suffering from chronic infectious diseases.

Induction of long-term protective effects against heterologous challenge in SIVhu-infected macaques.

A group of three rhesus macaques were inoculated with SIV isolated from a human (SIVhu) accidentally exposed and infected with SIVsm. Extensive sequence analyses of SIVhu obtained from the human and macaques following infection indicated the presence of truncated nef. Not only did nef fail to repair itself in vivo postinfection (p.i.), but instead, further mutations added additional stop codons with increasing time p.i. Infection of these animals was associated with minimal acute viral replication, followed by undetectable plasma viral loads and only intermittent PCR detection up to 5 years p.i. The three SIVhu infected and three control monkeys were then challenged with the heterologous highly pathogenic SHIV89.6p. All three controls became infected and showed rapid declines in peripheral CD4(+) lymphocytes, disease, and death at 10 and 32 weeks p.i., respectively. In contrast, all three animals previously infected with SIVhu are healthy and exhibit stable CD4(+) lymphocyte levels and undetectable plasma viral loads at >20 months post-SHIV89. 6p challenge. Only transient, low levels of SHIV replication were noted in these animals. Whereas responses to SIVgag/pol were noted, no evidence for SIV/SHIV envelope cross-reactivity was detected by antibody or CTL analyses, suggesting that the protective immune mechanisms to the heterologous challenge isolate were most likely not directed to envelope but rather to other viral determinants.

Efficacy of postexposure prophylaxis after intravaginal exposure of pig-tailed macaques to a human-derived retrovirus (human immunodeficiency virus type 2).

Postexposure prophylaxis (PEP) after intravaginal exposure to human immunodeficiency virus (HIV) was investigated using the HIV type 2 (HIV-2)/pig-tailed macaque transmission model. PEP for 28 days with the reverse transcriptase inhibitor (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA; tenofovir) was initiated 12 to 72 h following HIV-2 exposure. Systemic infection was not evident in the 12- and 36-h groups, as defined by plasma viremia, cell-associated provirus, antibody responses, and lymph node virus. Breakthrough infection in the 72-h group was detected at week 16 post-virus exposure. These results demonstrate for the first time using a vaginal transmission model that early intervention after high-risk sexual exposures may prevent infection.

Presence of diverse human immunodeficiency virus type 1 viral variants in Cameroon.

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.

Failure of routine HIV-1 tests in a case involving transmission with preseroconversion blood components during the infectious window period.

CONTEXT: Current screening practices for blood donations have been successful in reducing human immunodeficiency virus (HIV) transmission through receipt of contaminated blood products. However, HIV-infected blood donations made prior to seroconversion and before high levels of viral replication occur could test negative using both serologic antigen and antibody tests. Testing based on nucleic acid amplification (NAT) is being implemented to screen for HIV-infected blood donated during this period, yet the issue of single vs minipool donation screening remains unresolved. OBJECTIVES: To determine HIV-1 genetic linkage between virus in 2 HIV-1-infected recipients of blood components and virus in the donor, who was HIV antigen and antibody negative at the time of donation; to screen the blood donor's plasma with HIV NAT assays, including those currently proposed for use in US blood donation screening. DESIGN AND SETTING: Case study conducted in October 1997 involving the Communicable Disease Centre, Singapore General Hospital, and the Singapore Blood Transfusion Service, Singapore. SUBJECTS: The blood donor and the 2 recipients of donor platelets and red blood cells. MAIN OUTCOME MEASURES: Genetic analysis of the HIV-1 p17 coding region of gag and the C2V5 region of env to determine the genetic relatedness of virus from the donor and recipients; reactivity in quantitative and qualitative assays, and reactivity in donor screening HIV NAT assays in single donation and minipool screening contexts. RESULTS: Direct DNA sequencing demonstrated identical HIV-1 subtype E viral sequences in the donor and recipients. Based on comparisons of a qualitative and quantitative assay for HIV-1 RNA levels, a low level of viremia (range, 5-39 copies/mL in plasma) was estimated to be in the donor's undiluted blood at the time of donation. Additional testing using donor-screening NAT assays showed consistent detection of HIV RNA in the undiluted donor plasma whereas detection was inconsistent at the 1:16 and 1:24 dilution levels currently used in minipool screening of blood donations in the United States. CONCLUSIONS: Transmission of HIV from a blood donor to a platelet recipient and a red blood cell recipient occurred in the preseroconversion infectious window period. The viral load in the implicated donation was estimated to be less than 40 copies/mL of plasma. Current US minipool HIV NAT screening protocols may not be sufficiently sensitive to detect all infectious window-period donations. JAMA. 2000;284:210-214