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

Abstract

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 domesticus ticks were orally exposed to a high titer of MAL. At 3 weeks postinoculation (p.i.), <25% of the ticks contained detectable virus, with viral 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 growth defect, O. porcinus porcinus ticks were orally exposed to MAL and assayed at regular intervals p.i. Whole-tick viral titers dramatically declined (>1,000-fold) between 2 and 6 days p.i., and by 18 days p.i., viral titers were below the detection limit. In contrast, viral titers of ticks orally exposed to a tick-competent ASFV isolate, Pretoriuskop/96/4/1 (Pr4), increased 10-fold by 10 days p.i. and 50-fold by 14 days p.i. Early viral gene expression, but not extensive late gene expression or viral DNA synthesis, was 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 cytopathology of phagocytic midgut epithelial cells. To determine if viral replication was restricted only in the midgut epithelium, parenteral inoculations into the hemocoel were performed. With inoculation by this route, a persistent infection was established although a delay in generalization of MAL was detected and viral titers in most tissues were typically 10- to 1,000-fold lower than those of ticks injected with Pr4. MAL was detected in both the salivary secretion and coxal fluid following feeding but less frequently and at a lower titer compared to Pr4. Transovarial transmission of MAL was not detected after two gonotrophic cycles. Ultrastructural analysis demonstrated that, when injected, MAL replicated in a number of cell types but failed to replicate in midgut epithelial cells. In contrast, ticks injected with Pr4 had replicating virus in midgut epithelial cells. Together, these results indicate that MAL replication is restricted in midgut epithelial cells. This finding demonstrates the importance of viral replication in the midgut for successful ASFV infection of the arthropod host.

FIG. 1

Viral replication in O. porcinus porcinus ticks following oral exposure. Ticks were exposed by membrane feeding. The inoculum contained MAL or Pr4 diluted in either heparinized pig blood (A) or fetal bovine serum in place of pig blood (B). Immediately postfeeding, a portion of each group was killed by momentary freezing in liquid nitrogen. At the indicated times p.i., individual ticks (n = 4) from each group were ground in cell culture medium and titrations were performed. The values are mean titers ± the standard errors of the means.

FIG. 2

IHC and ISH analysis of midgut epithelial cells. Ticks were exposed by membrane feeding on an inoculum containing either MAL (A, C, and E) or Pr4 (B, D, and E). Analysis for ASFV protein p30 (A and B) or p72 (C and D) or ASFV DNA (E and F) was performed at 6 (A and B) or 11 (C, D, E, and F) days p.i. H, hemocoel; L, midgut lumen.

FIG. 3

ASFV in an O. porcinus porcinus midgut. Ticks were exposed by membrane feeding. The inoculum contained Pr4 (A and C) or MAL (B and D). Analysis was performed 6 days p.i. (A) Virus factory (VF) in a midgut epithelial cell from a Pr4-exposed tick. (B) Virus factory in a midgut epithelial cell from an MAL-exposed tick. (C) Higher magnification of a virus factory from a Pr4-exposed tick. (D) Higher magnification of a virus factory from an MAL-exposed tick. N, nucleus; L, midgut lumen; NF, nucleus free in the midgut lumen.

FIG. 3

ASFV in an O. porcinus porcinus midgut. Ticks were exposed by membrane feeding. The inoculum contained Pr4 (A and C) or MAL (B and D). Analysis was performed 6 days p.i. (A) Virus factory (VF) in a midgut epithelial cell from a Pr4-exposed tick. (B) Virus factory in a midgut epithelial cell from an MAL-exposed tick. (C) Higher magnification of a virus factory from a Pr4-exposed tick. (D) Higher magnification of a virus factory from an MAL-exposed tick. N, nucleus; L, midgut lumen; NF, nucleus free in the midgut lumen.

FIG. 4

Viral replication in O. porcinus porcinus ticks following intrahemocoelic (intrahemo.) or oral inoculation (inoc.). Ticks were infected by intrahemocoelic injection of either Pr4 or MAL or by feeding on a pig infected with Pr4. Individual ticks (n = 4) from each group were dissected at the indicated times p.i., and tissue viral titers were determined. Total-tick viral titers represent the sum of all dissected and undissected tissues. The values are mean titers ± the standard errors of the means.

FIG. 5

ASFV in an O. porcinus porcinus midgut. Analysis was performed 14 weeks after intrahemocoelic inoculation. (A) Mature virions (arrow) budding from a connective tissue cell adjacent to the basal lamina (BL) of the midgut in an MAL-injected tick. (B) Virus factory (VF) in a midgut epithelial cell from a Pr4-injected tick. H, hemocoel; L, lumen; M, muscle; N, nucleus.