African swine fever virus replication in the midgut epithelium is requiredfor infection of Ornithodoros ticks

Although the Malawi Lil20/1 (MAL) strain of African swine fever virus (ASFV ) was isolated from Ornithodoros sp. ticks, our attempts to experimentally infect ticks by feeding them this strain failed. Ten different collections of Ornithodorus porcinus porcinus ticks and one collection of O. porcinus d omesticus ticks were orally exposed to a high titer of MAL. At 3 weeks post inoculation (p.i.), <25% of the ticks contained detectable virus, with vira l titers of <4 log(10) 50% hemadsorbing doses/ml. Viral titers declined to undetectability in >90% of the ticks by 5 weeks p.i. To further study the g rowth defect, O. porcinus porcinus ticks were orally exposed to MAL and ass ayed at regular intervals p.i. Whole-tick viral titers dramatically decline d (>1,000-fold) between 2 and 6 days p.i., and by 18 days p.i., viral titer s were below the detection limit. In contrast, viral titers of ticks orally exposed to a tick-competent ASFV isolate, Pretoriuskop/96/4/1 (Pr4), incre ased 10-fold by 10 days p.i. and 50-fold by 14 days p.i. Early viral gene e xpression, but not extensive late gene expression or viral DNA synthesis, w as detected in the midguts of ticks orally exposed to MAL. Ultrastructural analysis demonstrated that progeny virus was rarely present in ticks orally exposed to MAL and, when present, was associated with extensive cytopathol ogy of phagocytic midgut epithelial cells. To determine if viral replicatio n was restricted only in the midgut epithelium, parenteral inoculations int o the hemocoel were performed. With inoculation by this route, a persistent infection was established although a delay in generalization of MAL was de tected and viral titers in most tissues were typically 10- to 1,000-fold lo wer than those of ticks injected with Pr4. MAL was detected in both the sal ivary secretion and coral fluid following feeding but less frequently and a t a lower titer compared to Pr4. Transovarial transmission of MAL was not d etected after two gonotrophic cycles. Ultrastructural analysis demonstrated that, when injected, MAL replicated in a number of cell types but failed t o replicate in midgut epithelial cells. In contrast, ticks injected with Pr 4 had replicating virus in midgut epithelial cells. Together, these results indicate that MAL replication is restricted in midgut epithelial cells. Th is finding demonstrates the importance of viral replication in the midgut f or successful ASFV infection of the arthropod host.