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To assess the ability of the African Hut Tampan, Ornithodoros moubata, to mechanically transmit HIV-1 and to re-appraise HIV-1 infectivity in an arthropod cell line at 28 and 35 °C.To evaluate HIV-1 transmission by O. moubata, as determined by HIV-1 survival ‘blood-meal’ size and interval between feeds, various tick developmental stages were allowed to feed on a heavily infected lymphoblast-rich blood-meal containing HIV-1BRU in an in vitro feeding chamber.Blood-meal regurgitation was evaluated using 51Cr-labelled human erythrocytes, and human lymphoblast survival in ticks using Trypan blue. HIV-1 survival in ticks was evaluated by reverse transcriptase activity in tick homogenates cocultured with CEM lymphoblasts. Polymerase chain reaction and Southern blot analysis were used to detect proviral HIV-1 in arthropod cells in vitro.HIV-1Bru remained viable for up to 10 days within O. moubata adults. This is the longest recorded survival of HIV in an arthropod. In agreement with other studies, O. moubata regurgitated part of its previous blood-meal into the feeding lesion. Human CEM lymphoblasts partially survived for up to 7 days at 28 and 35°C inside O. moubata's digestive tract. The blood-meal of adult female ticks was as high as 240 μ (approximately 70 times more than a mosquito), while the most likely potential mechanical vectors (fourth- and fifth-stage nymphs) ingested an average of 39 μ (maximum, 73 μl), with some ticks re-feeding as early as 14 days postfeed in the absence of a moult. Shortcomings associated with the experimental protocol suggest that HIV survival within O. moubata may reach 14 days following natural infection, or that ticks might re-feed earlier. Although HIV-1BRU and HIV-1NDK were unable to replicate at 28 and 35°C in CD4– Aedes albopictus C6/36 mosquito cells, HIV-1NDK was detected in its proviral form.Our investigations showed that mechanical transmission of HIV-1 by O. moubata is unlikely to occur in the laboratory. This may not be the situation under field conditions.