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.     

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 β-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.     

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.     

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.     

Cross-species transmission of simian foamy virus to humans in rural Gabon, Central Africa

In order to characterize simian foamy retroviruses (SFVs) in wild-born nonhuman primates (NHPs) in Gabon and to investigate cross-species transmission to humans, we obtained 497 NHP samples, composed of 286 blood and 211 tissue (bush meat) samples. Anti-SFV antibodies were found in 31 of 286 plasma samples (10.5%). The integrase gene sequence was found in 38/497 samples, including both blood and tissue samples, with novel SFVs in several Cercopithecus species. Of the 78 humans, mostly hunters, who had been bitten or scratched by NHPs, 19 were SFV seropositive, with 15 cases confirmed by PCR. All but one were infected with ape SFV. We thus found novel SFV strains in NHPs in Gabon and high cross-species transmission of SFVs from gorilla bites.     

Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections.     

Cholesterol is required for infection by Semliki Forest virus

Semliki Forest virus (SFV) and many other enveloped animal viruses enter cells by a membrane fusion reaction triggered by the low pH within the endocytic pathway. In vitro, SFV fusion requires cholesterol in the target membrane, but the role of cholesterol in vivo is unknown. In this paper, the infection pathway of SFV was studied in mammalian and inset cells substantially depleted of sterol. Cholesterol-depleted cells were unaltered in their ability to bind, internalize, and acidify virus, but were blocked in SFV fusion and subsequent virus replication. Depleted cells could be infected by the cholesterol-independent vesicular stomatitis virus, which also enters cells via endocytosis and low pH-mediated fusion. The block in SFV infection was specifically reversed by cholesterol but not by cholestenone, which lacks the critical 3 beta-hydroxyl group. Cholesterol thus is central in the infection pathway of SFV, and may act in vivo to modulate infection by SFV and other pathogens.     

Interspecies transmission of simian foamy virus in a natural predator-prey system

Simian foamy viruses (SFV) are ancient retroviruses of primates and have coevolved with their host species for as many as 30 million years. Although humans are not naturally infected with foamy virus, infection is occasionally acquired through interspecies transmission from nonhuman primates. We show that interspecies transmissions occur in a natural hunter-prey system, i.e., between wild chimpanzees and colobus monkeys, both of which harbor their own species-specific strains of SFV. Chimpanzees infected with chimpanzee SFV strains were shown to be coinfected with SFV from colobus monkeys, indicating that apes are susceptible to SFV superinfection, including highly divergent strains from other primate species.     

A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

The replicase protein nsP2 of Semliki Forest virus (SFV) has a ⁶⁴⁸RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.     

Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells.

We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the ts1 mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or ts1 mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV.     

Novel mutant Semliki Forest virus vectors: gene expression and localization studies in neuronal cells

Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology.     

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

Background

Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans.

Results

We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time.

Conclusion

Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.     

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.     

Replicative Intermediate of an Arbovirus

One hour after infection of chick fibroblasts with Semliki Forest virus (SFV), a viral ribonucleic acid (RNA) structure is present which has many of the properties described for the replicative intermediate of several RNA bacteriophages. These properties include a polydisperse nature on sucrose density gradient analysis, ribonuclease resistance, a variation in sedimentation pattern associated with changes in salt concentration, and recovery of infectious viral RNA upon denaturation. Most of the replicative intermediate present in SFV infection appears to be membrane-associated.     

Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus?

Murine F9 and PCC4 teratoma cells do not express H-2 major transplantation antigens according to virus-specific T-lymphocyte cytotoxic or serological assays. However, such cells can be infected with and readily replicate many types of viruses (coxsackie B 3, mouse hepatitis, Sindbis, Semliki Forest [SFV], lymphocytic choriomeningitis, Pichinde, vesicular stomatitis, herpes simplex type 1) to the same extent as do murine F12 teratoma cells and mouse embryo fibroblasts, all of which express the H-2 determinants. In contrast, F9 and PCC4 cells are not productively infected with murine cytomegalovirus, whereas F12 and mouse embryo fibroblast cells are. In addition to replicating in H-2-negative murine teratoma cells, SFV replicates in H-2-negative murine lymphoblastoid cells. The ability of SFV to infect cells without H-2 antigens and then to effect viral antigenic expression in the cells' cytoplasm and on their surface with similar kinetics and in equivalent amounts as cells with H-2 antigens indicates that the H-2 receptor is not needed for SFV infection. Daudi cells, which lack HLA antigens, block the replication of SFV. This occurs at some point after receptor binding, as demonstrated by diminished viral mRNA. In addition, a possible membrane defect precludes viral exit in Daudi cells transfected with SFV infectious RNA. These results indicate that a cell's possession of H-2 antigens is not a requirement for SFV infection and that major histocompatibility complex antigens are not specific receptors for this virus.