New World simian foamy virus infections in vivo and in vitro

Abstract

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.

FIG 1

Reactivity of Old and New World monkey plasma to SFVspm and SFVmac. Cell lysates were generated from uninfected HT1080 cells (UN), cells infected with SFVmac (MAC), or cells infected with SFVspm (SPM). (A) Western blot assays were performed using plasma (1:200) from four different squirrel monkeys (SQM 28, 2120, 2809, and 4028) and an anti-GAPDH antibody (1:3,000). (B) Western blot assays were performed using plasma (1:200) from a macaque known to be infected with SFVmac (MBG 35); an anti-GAPDH antibody was also used.

FIG 2

Nested PCR detection of SFV DNA in New World monkey and human blood. (A) A set of nested PCR primers were designed to specifically amplify a region of the SFV pol gene from NWM. Alignment of available New World SFV sequences from marmoset, spider, and squirrel monkeys allowed the design of primers in highly conserved regions. The red, lowercase letters indicate nonconsensus nucleotides. The nested PCR protocol amplified a 978-bp DNA fragment in the first round of PCR and a 403-bp DNA fragment in the second round. (B to E) Nested PCRs were performed, and second-round PCR products were visualized using gel electrophoresis and ethidium bromide staining. (B) Sensitivity of the NWM Pol primers was assessed by using known amounts of the SFVspm pol gene in the plasmid pSFVsqu3′ added to 200 ng of genomic DNA isolated from uninfected HT1080 cells. Samples included a DNA molecular size marker (lane 1), a no-DNA control (lane 2), and 10 (lanes 3 and 4), 5 (lanes 5 and 6), 1 (lanes 7 and 8), or no (lanes 9 and 10) copy equivalents of SFVsqu3′ mixed with 200 ng of HT1080 genomic DNA. (C) The ability of the NWM pol primers to detect SFV DNA extracted from infected cells in culture and from blood of New World monkeys was examined. Samples included a DNA molecular size marker (lane 1); no-template control (lane 2); 10 (lane 3), 5 (lane 4), or 1 (lane 5) copy equivalents of the plasmid pSFVsqu3′; 200 ng of genomic DNA extracted from uninfected (lane 6); SFVsqu-infected (lane 7); SFVspm-infected (lane 8) or SFVmar-infected (lane 9) Cf2Th cells; 200 ng of genomic DNA extracted from the whole blood of captive squirrel monkeys 2120 (lane 10), 2809 (lane 11), and 4028 (lane 12) and wild monkeys from Costa Rica: Howler monkey AP170 (lane 13) and AP171 (lane 14), capuchin CC74 (lane 15), and squirrel monkey SO39 (lane 16). (D and E) The presence of New and Old World SFV DNA in the blood of antibody-positive APH individuals was determined. (D) Samples included a DNA molecular size marker (lane 1), a no-template control (lane 2), or 10 (lane 3), 5 (lane 4), or 1 (lane 5) copy equivalents of the plasmid pSFVsqu3′. (E) Additional samples for pSFV-1 included 200 ng of genomic DNA extracted from whole blood of APH 3 (lane 6), APH 10 (lane 7), APH 25 (lane 8), APH 48 (lane 9), APH 71 (lane 10), APH 75 (lane 11), APH 84 (lane 12), and APH 111 (lane 13). NWM SFV pol primers were used in panel D; previously described primers specific to OW SFV pol (55) were used in panel E.

FIG 3

Reactivity of sera from humans occupationally exposed to SFVspm and SFVmac. Cell lysates were generated from uninfected HT1080 cells (UN), cells infected with SFVmac (MAC), or cells infected with SFVspm (SPM). Western blot assays were performed using plasma (1:20) collected from primatologists (APH) with documented exposures to NWM; an anti-GAPDH antibody (1:3,000) was also used. Representative Western blots are shown for the 8 of the 116 APH samples that were seropositive (APH 3, 10, 25, 48, 71, 75, 84, and 111) in addition to a Western blot showing results with plasma (1:20) from an individual with no known exposures to nonhuman primates (NHS) and an individual from Bangladesh known to be infected with SFVmac only (BGH 63).

FIG 4

Visualization of New World SFV-infected NFAB cells. The newly described NFAB indicator cells were infected with SFVsqm, SFVspm, SFVmar, or medium containing no virus. At 48 h postinfection, cells were fixed, incubated in X-Gal staining solution, and visualized at 200× magnification with an inverted microscope.