Simian Foamy Virus Transmission from Apes to Humans, Rural Cameroon

Bites from apes efficiently transmit the foamy virus to humans in natural settings in central Africa.

Publisher: CDC; Journal: Emerging Infectious Diseases Article Type: Research; Volume: 13; Issue: 9; Year: 2007; Article ID: 06-1162 DOI: 10.3201/eid1309.061162; TOC Head: Research indeterminate samples were then screened with another WB assay using, as viral antigen, a cellular lysate of BHK-21 cell line infected with a Cercopithecus SFV strain isolated from the AG16 case. For both tests, plasma samples were tested at a 1:100 dilution. Concerning the dried blood spot ("whatman samples"), a punch of approximately 1 cm in diameter was diluted in 1 ml of PBS and tested at a 1:8 dilution. WB seropositivity for foamy viruses was defined as the presence of a clear reactivity to the Gag doublet of 70 and 74 KDa for the Cpz assay and of 68KDa and 72 KDa for the monkey assay.

Virus isolation
Virus isolation was performed in two samples (AG15 and AG16) showing strong WB seropositivity, as previously described (14,34,37,50). Briefly, BHK-21 cells were maintained in DMEM medium supplemented with 5% fetal calf serum (FCS) and antibiotics. Fresh blood samples were collected in EDTA tubes and PBMCs (Peripheral Blood Mononuclear Cells) were isolated on a Ficoll-Hypaque gradient. PBMCs were then maintained for 2 days in RPMI medium containing 20% FCS, antibiotics and phytohemagglutinin (PHA) at 3 µg/ml or Protein A at 0,1 µg/ml and further stimulated with IL-2 (100U/ml). After 2 days of stimulation, PBMCs were co-cultivated with BHK-21 cells. Cultures were checked daily for syncytial cytopathic effect (CPE) typical of FV infection.
For transmission electron microscopy, cells were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in 0.15 M cacodylate buffer complemented with MgCl 2 , CaCl 2 and sucrose at 0.1M. After 2 days at 4°C, the filters were washed during 2 hours in cacodylate buffer and treated with a 1% osmium teroxide solution and 1% potassium ferrocyanide for 1 hour at room temperature. Cells were dehydrated in ethanol and embedded in epoxy resin at 60°C for 48hrs.
Ultrathin sections were obtained on a microtome Leica ultracut UCT. Sections were then examined in a Jeol 1200 EX electron microscope.

Indirect immunofluorescence
An indirect IF assay was performed on co-cultivated cells at 35 days post-infection. For the antibody positive control, we used, as the primary antibody, a serum derived from rabbit experimentally infected with a chimpanzee SFV strain; the secondary antibody was fluoresceinconjugated goat anti-rabbit diluted at 1:500. Cells were then mounted with DAPI-containing mounting medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Integrase PCR products were purified, cloned in a vector, and sequenced using the BigDye terminator cycle kit and an ABI 3100 automated sequencer (Applied Biosystem).
To determine the sensibility of our PCR assay, DNA from a cell line (HFV-2) containing 2 copies of integrated DNA/cell was extracted and measured by spectrophotometer. Serial dilutions of this DNA ranging from 1 μg (i.e. 150000 cells) to 0,01 ng were amplified in nested PCR for the integrase gene and in simple PCR (50 cycles) for the ß globin gene (Figure 1 supp.,   lanes 1-7). For the integrase PCR, each dilution was diluted in 100 ng of SFV negative DNA; the last positive dilution for integrase PCR (lane 7) corresponds to 1,5 cells or 3 copies of HFV. The last positive dilution for the ß globin PCR (lane 7) corresponds to 1,5 cells or 3 copies of ß globin. It means that the sensibility of our PCR ranges between 1-10 copies.