Neutralization of zoonotic retroviruses by human antibodies: Genotype-specific epitopes within the receptor-binding domain from simian foamy virus.

Infection with viruses of animal origin pose a significant threat to human populations. Simian foamy viruses (SFVs) are frequently transmitted to humans, in which they establish a life-long infection, with the persistence of replication-competent virus. However, zoonotic SFVs do not induce severe disease nor are they transmitted between humans. Thus, SFVs represent a model of zoonotic retroviruses that lead to a chronic infection successfully controlled by the human immune system. We previously showed that infected humans develop potent neutralizing antibodies (nAbs). Within the viral envelope (Env), the surface protein (SU) carries a variable region that defines two genotypes, overlaps with the receptor binding domain (RBD), and is the exclusive target of nAbs. However, its antigenic determinants are not understood. Here, we characterized nAbs present in plasma samples from SFV-infected individuals living in Central Africa. Neutralization assays were carried out in the presence of recombinant SU that compete with SU at the surface of viral vector particles. We defined the regions targeted by the nAbs using mutant SU proteins modified at the glycosylation sites, RBD functional subregions, and genotype-specific sequences that present properties of B-cell epitopes. We observed that nAbs target conformational epitopes. We identified three major epitopic regions: the loops at the apex of the RBD, which likely mediate interactions between Env protomers to form Env trimers, a loop located in the vicinity of the heparan binding site, and a region proximal to the highly conserved glycosylation site N8. We provide information on how nAbs specific for each of the two viral genotypes target different epitopes. Two common immune escape mechanisms, sequence variation and glycan shielding, were not observed. We propose a model according to which the neutralization mechanisms rely on the nAbs to block the Env conformational change and/or interfere with binding to susceptible cells. As the SFV RBD is structurally different from known retroviral RBDs, our data provide fundamental knowledge on the structural basis for the inhibition of viruses by nAbs. Trial registration: The study was registered at www.clinicaltrials.gov: https://clinicaltrials.gov/ct2/show/NCT03225794/.

An Immunodominant and Conserved B-Cell Epitope in the Envelope of Simian Foamy Virus Recognized by Humans Infected with Zoonotic Strains from Apes.

Cross-species transmission of simian foamy viruses (SFVs) from nonhuman primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently nonpathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N96-V110 located in the leader peptide, gp18LP Forty-three variant peptides with truncations, alanine substitutions, or amino acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old World monkey SFV species. The magnitude of binding to peptide N96-V110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodominant B-cell epitope recognized by humans following zoonotic SFV infection.IMPORTANCE Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. SFVs can be transmitted to humans, in whom they establish persistent infection, like the simian lenti- and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 and human T-lymphotropic virus type 1. This is the first identification of an SFV-specific B-cell epitope recognized by human plasma samples. The immunodominant epitope lies in gp18LP, probably at the base of the envelope trimers. The NHP species the most genetically related to humans transmitted SFV strains that induced the strongest antibody responses. Importantly, this epitope is well conserved across SFV species that infect African and Asian NHPs.

Isolation and sequence analysis of a novel rhesus macaque foamy virus isolate with a serotype-1-like env.

SFVmmu-DPZ9524 represents the third completely sequenced rhesus macaque simian foamy virus (SFV) isolate, alongside SFVmmu_K3T with a similar SFV-1-type env, and R289HybAGM with a SFV-2-like env. Sequence analysis demonstrates that, in gag and pol, SFVmmu-DPZ9524 is more closely related to R289HybAGM than to SFVmmu_K3T, which, outside of env, is more similar to a Japanese macaque isolate than to the other two rhesus macaque isolates SFVmmu-DPZ9524 and R289HybAGM. Further, we identify bel as another recombinant locus in R289HybAGM, confirming that recombination contributes to sequence diversity in SFV.

A Seminomadic Population in Bangladesh with Extensive Exposure to Macaques Does Not Exhibit High Levels of Zoonotic Simian Foamy Virus Infection.

Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.

Cocirculation of Two env Molecular Variants, of Possible Recombinant Origin, in Gorilla and Chimpanzee Simian Foamy Virus Strains from Central Africa.

UNLABELLED: Simian foamy virus (SFV) is a ubiquitous retrovirus in nonhuman primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory has identified more than 50 hunters from central Africa infected with zoonotic SFVs. Analysis of the complete sequences of five SFVs obtained from these individuals revealed that env was the most variable gene. Furthermore, recombinant SFV strains, some of which involve sequences in the env gene, were recently identified. Here, we investigated the variability of the env genes of zoonotic SFV strains and searched for possible recombinants. We sequenced the complete env gene or its surface glycoprotein region (SU) from DNA amplified from the blood of (i) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee foamy virus (FV) strain and (ii) 1 gorilla and 3 infected chimpanzees living in the same areas as these hunters. Phylogenetic analyses revealed the existence of two env variants among both the gorilla and chimpanzee FV strains that were present in zoonotic and NHP strains. These variants differ greatly (>30% variability) in a 753-bp-long region located in the receptor-binding domain of SU, whereas the rest of the gene is very conserved. Although the organizations of the Env protein sequences are similar, the potential glycosylation patterns differ between variants. Analysis of recombination suggests that the variants emerged through recombination between different strains, although all parental strains could not be identified. IMPORTANCE: SFV infection in humans is a great example of a zoonotic retroviral infection that has not spread among human populations, in contrast to human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). Recombination was a major mechanism leading to the emergence of HIV. Here, we show that two SFV molecular envelope gene variants circulate among ape populations in Central Africa and that both can be transmitted to humans. These variants differ greatly in the SU region that corresponds to the part of the Env protein in contact with the environment. These variants may have emerged through recombination between SFV strains infecting different NHP species.

An expanded search for simian foamy viruses (SFV) in Brazilian New World primates identifies novel SFV lineages and host age-related infections.

BACKGROUND: While simian foamy viruses have co-evolved with their primate hosts for millennia, most scientific studies have focused on understanding infection in Old World primates with little knowledge available on the epidemiology and natural history of SFV infection in New World primates (NWPs). To better understand the geographic and species distribution and evolutionary history of SFV in NWPs we extend our previous studies in Brazil by screening 15 genera consisting of 29 NWP species (140 monkeys total), including five genera (Brachyteles, Cacajao, Callimico, Mico, and Pithecia) not previously analyzed. Monkey blood specimens were tested using a combination of both serology and PCR to more accurately estimate prevalence and investigate transmission patterns. Sequences were phylogenetically analyzed to infer SFV and host evolutionary histories. RESULTS: The overall serologic and molecular prevalences were 42.8 and 33.6 %, respectively, with a combined assay prevalence of 55.8 %. Discordant serology and PCR results were observed for 28.5 % of the samples, indicating that both methods are currently necessary for estimating NWP SFV prevalence. SFV prevalence in sexually mature NWPs with a positive result in any of the WB or PCR assays was 51/107 (47.7 %) compared to 20/33 (61 %) for immature animals. Epidemiological analyses revealed an increase in SFV prevalence with age in captive Cebus monkeys. Phylogenetic analysis identified novel SFVs in Cacajao, Leontopithecus, and Chiropotes species that had 6-37 % nucleotide divergence to other NWP SFV. Comparison of host and SFV phylogenies showed an overall cospeciation evolutionary history with rare ancient and contemporaneous host-switching for Saimiri and Leontopithecus and Cebus xanthosternos, respectively. CONCLUSIONS: We identified novel SFV in four neotropical monkey genera in Brazil and demonstrate that SFV prevalence increases with age in Cebus monkeys. Importantly, our test results suggest that both molecular and serological screening are currently required to accurately determine infection with NWP SFV. Our study significantly expands knowledge of the epidemiology and natural history of NWP SFVs. The tools and information provided in our study will facilitate further investigation of SFV in NWPs and the potential for zoonotic infection with these viruses.

Wide distribution and ancient evolutionary history of simian foamy viruses in New World primates.

BACKGROUND: Although simian foamy viruses (SFV) are the only exogenous retroviruses to infect New World monkeys (NWMs), little is known about their evolutionary history and epidemiology. Previous reports show distinct SFVs among NWMs but were limited to small numbers of captive or wild monkeys from five (Cebus, Saimiri, Ateles, Alouatta, and Callithrix) of the 15 NWM genera. Other studies also used only PCR testing or serological assays with limited validation and may have missed infection in some species. We developed and validated new serological and PCR assays to determine the prevalence of SFV in blood specimens from a large number of captive NWMs in the US (n = 274) and in captive and wild-caught NWMs (n = 236) in Peruvian zoos, rescue centers, and illegal trade markets. Phylogenetic and co-speciation reconciliation analyses of new SFV polymerase (pol) and host mitochondrial cytochrome B sequences, were performed to infer SFV and host co-evolutionary histories. RESULTS: 124/274 (45.2 %) of NWMs captive in the US and 59/157 (37.5 %) of captive and wild-caught NWMs in Peru were SFV WB-positive representing 11 different genera (Alouatta, Aotus, Ateles, Cacajao, Callithrix, Cebus, Lagothrix, Leontopithecus, Pithecia, Saguinus and Saimiri). Seroprevalences were lower at rescue centers (10/53, 18.9 %) compared to zoos (46/97, 47.4 %) and illegal trade markets (3/7, 8/19, 42.9 %) in Peru. Analyses showed that the trees of NWM hosts and SFVs have remarkably similar topologies at the level of species and sub-populations suggestive of co-speciation. Phylogenetic reconciliation confirmed 12 co-speciation events (p < 0.002) which was further supported by obtaining highly similar divergence dates for SFV and host genera and correlated SFV-host branch times. However, four ancient cross-genus transmission events were also inferred for Pitheciinae to Atelidae, Cacajao to ancestral Callithrix or Cebus monkeys, between Callithrix and Cebus monkeys, and Lagothrix to Alouatta. CONCLUSIONS: We demonstrate a broad distribution and stable co-speciation history of SFV in NWMs at the species level. Additional studies are necessary to further explore the epidemiology and natural history of SFV infection of NWMs and to determine the zoonotic potential for persons exposed to infected monkeys in captivity and in the wild.

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins.

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross-loop base pair leaving a triloop and 3' bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross-loop base pair, also a 3' pyrimidine bulge and a G-C loop-closing base pair are primary determinants of PTL formation. By changing the G-C closing base pair into C-G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.

New World simian foamy virus infections in vivo and in vitro.

Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a ß-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence.

A unique spumavirus Gag N-terminal domain with functional properties of orthoretroviral matrix and capsid.

The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

A novel Bayesian method for detection of APOBEC3-mediated hypermutation and its application to zoonotic transmission of simian foamy viruses.

Simian Foamy Virus (SFV) can be transmitted from non-human primates (NHP) to humans. However, there are no documented cases of human to human transmission, and significant differences exist between infection in NHP and human hosts. The mechanism for these between-host differences is not completely understood. In this paper we develop a new Bayesian approach to the detection of APOBEC3-mediated hypermutation, and use it to compare SFV sequences from human and NHP hosts living in close proximity in Bangladesh. We find that human APOBEC3G can induce genetic changes that may prevent SFV replication in infected humans in vivo.

Identification of recombination in the envelope gene of simian foamy virus serotype 2 isolated from Macaca cyclopis.

The full-length sequence of simian foamy virus serotype 2 (SFVmcy-2), isolated from a Taiwanese macaque, was determined. SFVmcy-2 was highly related to SFV serotype 1 (SFVmcy-1), an isolate from the same species, except in the putative receptor binding domain (RBD) in env, which contained novel sequences related to SFV serotype 3 (SFVagm-3), isolated from an African green monkey. The results identify a potential region of neutralization in SFVs and demonstrate recombination between genetically divergent foamy viruses.

Simian foamy virus infection of rhesus macaques in Bangladesh: relationship of latent proviruses and transcriptionally active viruses.

Simian foamy viruses (SFV) are complex retroviruses that are ubiquitous in nonhuman primates (NHP) and are zoonotically transmitted to humans, presumably through NHP saliva, by licking, biting, and other behaviors. We have studied SFV in free-ranging rhesus macaques in Bangladesh. It has been previously shown that SFV in immunocompetent animals replicates to detectable levels only in superficial epithelial cells of the oral mucosa, although latent proviruses are found in most, if not all, tissues. In this study, we compare DNA sequences from latent SFV proviruses found in blood cells of 30 Bangladesh rhesus macaques to RNA sequences of transcriptionally active SFV from buccal swabs obtained from the same animals. Viral strains, defined by differences in SFV gag sequences, from buccal mucosal specimens overlapped with those from blood samples in 90% of animals. Thus, latent proviruses in peripheral blood mononuclear cells (PBMC) are, to a great extent, representative of viruses likely to be transmitted to other hosts. The level of SFV RNA in buccal swabs varied greatly between macaques, with increasing amounts of viral RNA in older animals. Evidence of APOBEC3-induced mutations was found in gag sequences derived from the blood and oral mucosa.

The impact of calibration and clock-model choice on molecular estimates of divergence times.

Phylogenetic estimates of evolutionary timescales can be obtained from nucleotide sequence data using the molecular clock. These estimates are important for our understanding of evolutionary processes across all taxonomic levels. The molecular clock needs to be calibrated with an independent source of information, such as fossil evidence, to allow absolute ages to be inferred. Calibration typically involves fixing or constraining the age of at least one node in the phylogeny, enabling the ages of the remaining nodes to be estimated. We conducted an extensive simulation study to investigate the effects of the position and number of calibrations on the resulting estimate of the timescale. Our analyses focused on Bayesian estimates obtained using relaxed molecular clocks. Our findings suggest that an effective strategy is to include multiple calibrations and to prefer those that are close to the root of the phylogeny. Under these conditions, we found that evolutionary timescales could be estimated accurately even when the relaxed-clock model was misspecified and when the sequence data were relatively uninformative. We tested these findings in a case study of simian foamy virus, where we found that shallow calibrations caused the overall timescale to be underestimated by up to three orders of magnitude. Finally, we provide some recommendations for improving the practice of molecular-clock calibration.

Evaluation and prediction of the HIV-1 central polypurine tract influence on foamy viral vectors to transduce dividing and growth-arrested cells.

Retroviral vectors are potent tools for gene delivery and various biomedical applications. To accomplish a gene transfer task successfully, retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle. However, very promising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells. It is hypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC) with nuclear import activity in growth-arrested cells, which is the characteristic for lentiviruses (HIV-1). In this process, the HIV-1 central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a "flap" element and is believed to be crucial for the subsequent double-stranded cDNA formation of all retroviral RNA genomes. In this study, the effects of the lentiviral cPPT element on the FV transduction potential in dividing and growth-arrested (G1/S phase) adenocarcinomic human alveolar basal epithelial (A549) cells are investigated by experimental and theoretical methods. The results indicated that the HIV-1 cPPT element in a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cells due to the absence of PICs with nuclear import activity.

Foamy virus envelope protein is a substrate for signal peptide peptidase-like 3 (SPPL3).

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.

An endogenous foamy virus in the aye-aye (Daubentonia madagascariensis).

We report the discovery and analysis of an endogenous foamy virus (PSFVaye) within the genome of the aye-aye (Daubentonia madagascariensis), a strepsirrhine primate from Madagascar. Phylogenetic analyses indicate that PSFVaye is divergent from all known simian foamy viruses, suggesting an association between foamy viruses and primates since the haplorrhine-strepsirrhine split. The discovery of PSFVaye indicates that primate foamy virus might be more broadly distributed than previously thought.

Genetic characterization of simian foamy viruses infecting humans.

Simian foamy viruses (SFVs) are retroviruses that are widespread among nonhuman primates (NHPs). SFVs actively replicate in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data on the genetic structure of such SFVs found in humans are available. In the framework of ongoing studies searching for SFV-infected humans in south Cameroon rainforest villages, we studied 38 SFV-infected hunters whose times of infection had presumably been determined. By long-term cocultures of peripheral blood mononuclear cells with BHK-21 cells, we isolated five new SFV strains and obtained complete genomes of SFV strains from chimpanzee (Pan troglodytes troglodytes; strains BAD327 and AG15), monkey (Cercopithecus nictitans; strain AG16), and gorilla (Gorilla gorilla; strains BAK74 and BAD468). These zoonotic strains share a very high degree of similarity with their NHP counterparts and have a high degree of conservation of the genetic elements important for viral replication. Interestingly, analysis of FV DNA sequences obtained before cultivation revealed variants with deletions in both the U3 region and tas that may correlate with in vivo chronicity in humans. Genomic changes in bet (a premature stop codon) and gag were also observed. To determine if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes. Our study reveals that natural polymorphism of SFV strains does exist at both the intersubspecies level (gag, bet) and the intrasubspecies (U3, tas) levels but does not seem to reflect a viral adaptation specific to zoonotic SFV strains.

Frequent and recent human acquisition of simian foamy viruses through apes' bites in central Africa.

Human infection by simian foamy viruses (SFV) can be acquired by persons occupationally exposed to non-human primates (NHP) or in natural settings. This study aimed at getting better knowledge on SFV transmission dynamics, risk factors for such a zoonotic infection and, searching for intra-familial dissemination and the level of peripheral blood (pro)viral loads in infected individuals. We studied 1,321 people from the general adult population (mean age 49 yrs, 640 women and 681 men) and 198 individuals, mostly men, all of whom had encountered a NHP with a resulting bite or scratch. All of these, either Pygmies (436) or Bantus (1085) live in villages in South Cameroon. A specific SFV Western blot was used and two nested PCRs (polymerase, and LTR) were done on all the positive/borderline samples by serology. In the general population, 2/1,321 (0.2%) persons were found to be infected. In the second group, 37/198 (18.6%) persons were SFV positive. They were mostly infected by apes (37/39) FV (mainly gorilla). Infection by monkey FV was less frequent (2/39). The viral origin of the amplified sequences matched with the history reported by the hunters, most of which (83%) are aged 20 to 40 years and acquired the infection during the last twenty years. The (pro)viral load in 33 individuals infected by a gorilla FV was quite low (<1 to 145 copies per 10(5) cells) in the peripheral blood leucocytes. Of the 30 wives and 12 children from families of FV infected persons, only one woman was seropositive in WB without subsequent viral DNA amplification. We demonstrate a high level of recent transmission of SFVs to humans in natural settings specifically following severe gorilla bites during hunting activities. The virus was found to persist over several years, with low SFV loads in infected persons. Secondary transmission remains an open question.