Local Anesthetic Systemic Toxicity: Ensuring Sustained Nursing Knowledge in a High-Volume Outpatient Surgery Center.

PURPOSE: Local anesthetic systemic toxicity (LAST) is a low-frequency, high-risk event that can occur within minutes of a patient receiving a local anesthetic. The goals of this project were to standardize LAST care management across an academic medical center and sustain an improvement in nurses' knowledge of how to recognize signs and symptoms of LAST and how to competently manage a LAST scenario. DESIGN: We used a quantitative design to accomplish the goals of the project. METHODS: Our interdisciplinary team developed a clinical practice guideline based on the LAST Checklist published by the American Society of Regional Anesthesia and Pain Medicine, and used a simulation scaffolded by multimodal education and system changes to ensure sustained knowledge. We measured improvement using a graded knowledge assessment as well as qualitative feedback. FINDINGS: Scores on the assessment increased from 4.76 to 6.34 (out of seven points) following the intervention and remained significantly higher than the baseline 9 months after the educational intervention (9-month score = 6.19, t = 2.99, P = .004). Nurses reported feeling more confident and knowledgeable following the intervention and requested to have regular sessions of the simulation. To sustain improvements, we developed a computer-based learning module. The module and simulation were integrated into nursing orientation and an annual competency. CONCLUSIONS: While standardizing LAST care in accordance with evidence-based guidance is critical to patient safety due to its infrequent occurrence, nurses should consider implementing simulation supplemented with multimodal education and system changes to ensure sustained knowledge.

Early antibody therapy can induce long-lasting immunity to SHIV.

Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans. In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56-177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4+) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 106 circulating CD4+ T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8ß monoclonal antibody to the controller animals led to a specific decline in levels of CD8+ T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8+ T-cell immunity able to durably suppress virus replication.

The Oldest Co-opted gag Gene of a Human Endogenous Retrovirus Shows Placenta-Specific Expression and Is Upregulated in Diffuse Large B-Cell Lymphomas.

Vertebrate genomes contain endogenous retroviruses (ERVs) that represent remnants of past germline infections by ancient retroviruses. Despite comprising 8% of the human genome, the human ERVs (HERVs) do not encode a replication competent retrovirus. However, some HERV genes have been co-opted to serve host functions, most notably the viral envelope-derived syncytins involved in placentation. Here, we identify the oldest HERV intact gag gene with an open reading frame, gagV1. Its provirus contains an intact env, envV1, and the first open reading frame found in an HERV gag leader, pre-gagV1, which encodes a novel protein. This HERV is linked to a related gag gene, gagV3, and these three genes all show patterns of evolutionary conservation in primates. gagV1 and pre-gagV1 orthologs are present in all simian primate lineages indicating that this HERV entered the germline of the common simian primate ancestor at least 43 Ma, whereas gagV3 is found in Old and New World monkeys. gagV1 and gagV3 have undergone recurrent gene conversion events and positive selection. Expression of gagV1, gagV3, and pre-gagV1 is restricted to the placenta in humans and macaques suggesting co-option for placenta-specific host functions. Transcriptomic analysis of human tumors also found upregulated levels of gagV1 transcripts in diffuse large B-cell lymphomas. These findings suggest that these HERV-V genes may be useful markers for the most common type of non-Hodgkin's lymphoma and that they may have contributed to the successive domestications of env and gag genes in eutherians involved in the ongoing ERV-driven evolution of the placenta.

A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges.

Despite the success of potent anti-retroviral drugs in controlling human immunodeficiency virus type 1 (HIV-1) infection, little progress has been made in generating an effective HIV-1 vaccine. Although passive transfer of anti-HIV-1 broadly neutralizing antibodies can protect mice or macaques against a single high-dose challenge with HIV or simian/human (SIV/HIV) chimaeric viruses (SHIVs) respectively, the long-term efficacy of a passive antibody transfer approach for HIV-1 has not been examined. Here we show, on the basis of the relatively long-term protection conferred by hepatitis A immune globulin, the efficacy of a single injection (20 mg kg(-1)) of four anti-HIV-1-neutralizing monoclonal antibodies (VRC01, VRC01-LS, 3BNC117, and 10-1074 (refs 9 - 12)) in blocking repeated weekly low-dose virus challenges of the clade B SHIVAD8. Compared with control animals, which required two to six challenges (median = 3) for infection, a single broadly neutralizing antibody infusion prevented virus acquisition for up to 23 weekly challenges. This effect depended on antibody potency and half-life. The highest levels of plasma-neutralizing activity and, correspondingly, the longest protection were found in monkeys administered the more potent antibodies 3BNC117 and 10-1074 (median = 13 and 12.5 weeks, respectively). VRC01, which showed lower plasma-neutralizing activity, protected for a shorter time (median = 8 weeks). The introduction of a mutation that extends antibody half-life into the crystallizable fragment (Fc) domain of VRC01 increased median protection from 8 to 14.5 weeks. If administered to populations at high risk of HIV-1 transmission, such an immunoprophylaxis regimen could have a major impact on virus transmission.

Reduced glucose-stimulated insulin secretion following a 1-wk IGF-1 infusion in late gestation fetal sheep is due to an intrinsic islet defect.

Insulin and insulin-like growth factor-1 (IGF-1) are fetal hormones critical to establishing normal fetal growth. Experimentally elevated IGF-1 concentrations during late gestation increase fetal weight but lower fetal plasma insulin concentrations. We therefore hypothesized that infusion of an IGF-1 analog for 1 wk into late gestation fetal sheep would attenuate fetal glucose-stimulated insulin secretion (GSIS) and insulin secretion in islets isolated from these fetuses. Late gestation fetal sheep received infusions with IGF-1 LR3 (IGF-1, n = 8), an analog of IGF-1 with low affinity for the IGF binding proteins and high affinity for the IGF-1 receptor, or vehicle control (CON, n = 9). Fetal GSIS was measured with a hyperglycemic clamp (IGF-1, n = 8; CON, n = 7). Fetal islets were isolated, and insulin secretion was assayed in static incubations (IGF-1, n = 8; CON, n = 7). Plasma insulin and glucose concentrations in IGF-1 fetuses were lower compared with CON (P = 0.0135 and P = 0.0012, respectively). During the GSIS study, IGF-1 fetuses had lower insulin secretion compared with CON (P = 0.0453). In vitro, glucose-stimulated insulin secretion remained lower in islets isolated from IGF-1 fetuses (P = 0.0447). In summary, IGF-1 LR3 infusion for 1 wk into fetal sheep lowers insulin concentrations and reduces fetal GSIS. Impaired insulin secretion persists in isolated fetal islets indicating an intrinsic islet defect in insulin release when exposed to IGF-1 LR3 infusion for 1 wk. We speculate this alteration in the insulin/IGF-1 axis contributes to the long-term reduction in ß-cell function in neonates born with elevated IGF-1 concentrations following pregnancies complicated by diabetes or other conditions associated with fetal overgrowth.NEW & NOTEWORTHY After a 1-wk infusion of IGF-1 LR3, late gestation fetal sheep had lower plasma insulin and glucose concentrations, reduced fetal glucose-stimulated insulin secretion, and decreased fractional insulin secretion from isolated fetal islets without differences in pancreatic insulin content.

IGF-1 infusion to fetal sheep increases organ growth but not by stimulating nutrient transfer to the fetus.

Insulin-like growth factor-1 (IGF-1) is an important fetal growth factor. However, the role of fetal IGF-1 in increasing placental blood flow, nutrient transfer, and nutrient availability to support fetal growth and protein accretion is not well understood. Catheterized fetuses from late gestation pregnant sheep received an intravenous infusion of LR3 IGF-1 (LR3 IGF-1; n = 8) or saline (SAL; n = 8) for 1 wk. Sheep then underwent a metabolic study to measure uterine and umbilical blood flow, nutrient uptake rates, and fetal protein kinetic rates. By the end of the infusion, fetal weights were not statistically different between groups (SAL: 3.260 ± 0.211 kg, LR3 IGF-1: 3.682 ± 0.183; P = 0.15). Fetal heart, adrenal gland, and spleen weights were higher (P < 0.05), and insulin was lower in LR3 IGF-1 (P < 0.05). Uterine and umbilical blood flow and umbilical uptake rates of glucose, lactate, and oxygen were similar between groups. Umbilical amino acid uptake rates were lower in LR3 IGF-1 (P < 0.05) as were fetal concentrations of multiple amino acids. Fetal protein kinetic rates were similar. LR3 IGF-1 skeletal muscle had higher myoblast proliferation (P < 0.05). In summary, LR3 IGF-1 infusion for 1 wk into late gestation fetal sheep increased the weight of some fetal organs. However, because umbilical amino acid uptake rates and fetal plasma amino acid concentrations were lower in the LR3 IGF-1 group, we speculate that animals treated with LR3 IGF-1 can efficiently utilize available nutrients to support organ-specific growth in the fetus rather than by stimulating placental blood flow or nutrient transfer to the fetus.NEW & NOTEWORTHY After a 1-wk infusion of LR3 IGF-1, late gestation fetal sheep had lower umbilical uptake rates of amino acids, lower fetal arterial amino acid and insulin concentrations, and lower fetal oxygen content; however, LR-3 IGF-1-treated fetuses were still able to effectively utilize the available nutrients and oxygen to support organ growth and myoblast proliferation.

Ancient Evolutionary Origin and Positive Selection of the Retroviral Restriction Factor Fv1 in Muroid Rodents.

The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirus L (MuERV-L) family of ERVs suggests that Fv1 was coopted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the genus Mus Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside the genus Mus We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1 Our analysis also revealed that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the genus Mus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together, these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs.IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, encoded by the Fv1 gene found in mice, was thought to have originated in the genus Mus In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum time of insertion into the genome of rodents of 45 million years for the ancestral progenitor of Fv1 While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular "arms race" in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been coevolving with unidentified retroviruses for millions of years.

Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia.

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9-14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5 weeks in some long-term chronically SHIV-infected animals with low CD4(+) T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Sequence Diversity, Intersubgroup Relationships, and Origins of the Mouse Leukemia Gammaretroviruses of Laboratory and Wild Mice.

UNLABELLED: Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies ofMus musculus Receptor usage and envelope (env) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions ofenvproduced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome.envvariations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region ofenv Outside theenvregion, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributedMussubspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. IMPORTANCE: Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution.

TRIM5α Resistance Escape Mutations in the Capsid Are Transferable between Simian Immunodeficiency Virus Strains.

TRIM5α polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5α-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5α restriction when it was passaged in rhesus macaques expressing restrictive TRIM5α alleles. Here we generated TRIM5α-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5α alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5α genotypes as efficiently as in macaques with permissive TRIM5α genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5α restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies. IMPORTANCE: Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5α alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5α genotype and thus will eliminate the confounding effects of TRIM5α in vaccine studies.

The Expression of Functional Vpx during Pathogenic SIVmac Infections of Rhesus Macaques Suppresses SAMHD1 in CD4+ Memory T Cells.

For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.

TRIM5α restriction affects clinical outcome and disease progression in simian immunodeficiency virus-infected rhesus macaques.

UNLABELLED: Tripartite motif-containing protein 5α (TRIM5α) is considered to be a potential target for cell-based gene modification therapy against human immunodeficiency virus type 1 (HIV-1) infection. In the present study, we used a relevant rhesus macaque model of infection with simian immunodeficiency virus from sooty mangabey (SIVsm) to evaluate the effect of TRIM5α restriction on clinical outcome. For macaques expressing a restrictive TRIM5 genotype, the disease outcomes of those infected with the wild-type TRIM-sensitive SIVsm strain and those infected with a virus with escape mutations in the capsid were compared. We found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, supporting the feasibility of exploiting TRIM5α as a target for gene therapy against HIV-1. Furthermore, we also found that preservation of memory CD4 T cells was associated with protection by TRIM5α restriction, suggesting memory CD4 T cells or their progenitor cells as an ideal target for gene modification. Despite the significant effect of TRIM5α restriction on survival, SIV escape from TRIM5α restriction was also observed; therefore, this may not be an effective stand-alone strategy and may require combination with other targets. IMPORTANCE: Recent studies suggest that it may be feasible not only to suppress viral replication with antiviral drugs but also potentially to eliminate or "cure" human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5α. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic, and some alleles are restrictive for primary SIVsm isolates, although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 on macaques. The present study evaluated these animals for long-term outcomes and found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, suggesting that this could be a valid gene therapy approach that could be adapted for HIV.

Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors.

UNLABELLED: African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo, while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4(+) T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4(+) T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals. IMPORTANCE: African green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptors in vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entry in vitro and may serve as entry coreceptors for SIVagm in vivo, since their mRNAs were detected in AGM memory CD4(+) T cells, the preferred target cells of SIV.

Characterization of simian immunodeficiency virus (SIV) that induces SIV encephalitis in rhesus macaques with high frequency: role of TRIM5 and major histocompatibility complex genotypes and early entry to the brain.

UNLABELLED: Although nonhuman primate models of neuro-AIDS have made tremendous contributions to our understanding of disease progression in the central nervous system (CNS) of human immunodeficiency virus type 1 (HIV-1)-infected individuals, each model holds advantages and limitations. In this study, in vivo passage of SIVsmE543 was conducted to obtain a viral isolate that can induce neuropathology in rhesus macaques. After a series of four in vivo passages in rhesus macaques, we have successfully isolated SIVsm804E. SIVsm804E shows efficient replication in peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) in vitro and induces neuro-AIDS in high frequencies in vivo. Analysis of the acute phase of infection revealed that SIVsm804E establishes infection in the CNS during the early phase of the infection, which was not observed in the animals infected with the parental SIVsmE543-3. Comprehensive analysis of disease progression in the animals used in the study suggested that host major histocompatibility complex class I (MHC-I) and TRIM5α genotypes influence the disease progression in the CNS. Taken together, our findings show that we have successfully isolated a new strain of simian immunodeficiency virus (SIV) that is capable of establishing infection in the CNS at early stage of infection and causes neuropathology in infected rhesus macaques at a high frequency (83%) using a single inoculum, when animals with restrictive MHC-I or TRIM5α genotypes are excluded. SIVsm804E has the potential to augment some of the limitations of existing nonhuman primate neuro-AIDS models. IMPORTANCE: Human immunodeficiency virus (HIV) is associated with a high frequency of neurologic complications due to infection of the central nervous system (CNS). Although the use of antiviral treatment has reduced the incidence of severe complications, milder disease of the CNS continues to be a significant problem. Animal models to study development of neurologic disease are needed. This article describes the development of a novel virus isolate that induces neurologic disease in a high proportion of rhesus macaques infected without the need for prior immunomodulation as is required for some other models.

TRIM5 alpha drives SIVsmm evolution in rhesus macaques.

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5(Q) was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5(TFP/TFP) or TRIM5(Cyp/TFP). Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5(TFP) restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5(Cyp). Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIM(TFP) alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.

Most rhesus macaques infected with the CCR5-tropic SHIV(AD8) generate cross-reactive antibodies that neutralize multiple HIV-1 strains.

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIV(AD8-EO)) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1(DH12) strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIV(AD8) stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIV(AD8)-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti-HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41-51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIV(AD8) macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains.

The avian XPR1 gammaretrovirus receptor is under positive selection and is disabled in bird species in contact with virus-infected wild mice.

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.

Emergence of gp120 V3 variants confers neutralization resistance in an R5 simian-human immunodeficiency virus-infected macaque elite neutralizer that targets the N332 glycan of the human immunodeficiency virus type 1 envelope glycoprotein.

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIVAD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.