Fig 1.
Influence of virus dose on infection and transmission rates.
Mosquitoes were allowed to feed on a suspension of bovine erythrocytes containing a low dose (LD, 105.3 TCID50/ml), medium dose (MD, 107.3 TCID50/ml) or high dose (HD, 109.3 TCID50/ml) of RVFV Clone 13 and were maintained for 21 days at 28°C. (A) Symbols represent virus titers in the bodies of the mosquitoes that were found virus positive after the incubation period. Means with SDs (error bars) and the detection limit of the virus isolation assay (dashed line) are indicated. (B) Infection and transmission rates. Asterisks indicate statistically significant differences (P<0.017) as determined using the Mann-Whitney test (panel A) or Fisher’s exact test (panel B).
Table 1.
Infection and dissemination rates in Cx. pipiens mosquitoes after oral exposure to Clone 13 or wild-type RVFV strain 35/74a.
Fig 2.
Vector competence of Cx. pipiens hatched from field-collected mosquito eggs.
Mosquitoes hatched from field-collected eggs were allowed to feed on a blood meal containing 109.3 TCID50/ml of Clone 13. After incubation for 14 days at 28°C, the titers in mosquito bodies (A) and infection and transmission rates (B) were determined.
Fig 3.
Comparing the vector competence of Cx. pipiens hatched from field-collected mosquito eggs for Clone 13 and wild-type strain 35/74.
Mosquitoes hatched from field-collected eggs were allowed to feed on a blood meal containing 108.0 TCID50/ml of Clone 13 or virulent strain 35/74. After incubation for 14 days at 28°C, the titers in mosquito bodies (A) and infection and transmission rates (B) were determined. Asterisks indicate statistically significant differences (P<0.05).
Fig 4.
Feeding of Cx. pipiens mosquitoes on lambs during RVFV viremia.
Two lambs were inoculated via intravenous route with the highly virulent 35/74 strain. Groups of 50 mosquitoes were allowed to feed on the lambs every following day. The method used to place containers on the lambs is depicted in panels A-C. Both lambs developed fever on DPI 2 (D). Lamb 1 () was euthanized on DPI 4, whereas lamb 2 (
) succumbed to the infection on DPI 3. E. Levels of viral RNA (dashed lines) and infectious virus (solid lines) in plasma samples of the lambs. F. Ratios of viral RNA and infectious virus during viremia.
Fig 5.
Transmission of RVFV from viremic lambs to Cx. pipiens mosquitoes.
Mosquitoes were allowed to feed on lambs at days 1–4 post infection (DPI). Virus titers in the bodies of the mosquitoes after the incubation period are shown in panels A (mosquitoes that fed on lamb 1) and B (mosquitoes that fed on lamb 2). Means with SDs and the detection limit of the virus isolation assay are indicated. The transmission rates are shown in panels C (mosquitoes that fed on lamb 1) and D (mosquitoes that fed on lamb 2). Asterisks indicate statistically significant differences (P<0.01) between the indicated group and the other groups as determined using the Mann-Whitney test (A and B) or Fisher’s exact test (C and D).
Fig 6.
Hematoxylin and eosin staining of unexposed (A) and mosquito-exposed (B) skin. Staining of mosquito-exposed skin reveals dilatation of the blood vessels with extensive haemorrhages in the dermis. Hydropic degeneration of keratinocytes in the epidermis, exocytosis of neutrophils and crust formation (green arrowhead). (C) Margination of neutrophils and thrombocytes in capillaries and venules (green arrowheads). (D) Influx of neutrophils (yellow arrowheads) and macrophages (green arrowheads) into the dermis. Macrophages show phagocytosis of erythrocytes (green arrowheads) and an apoptotic neutrophil (red arrowhead). Bar = 200 μm (A, B), 20 μm (C, D).
Fig 7.
Increased virus replication in the skin of lambs after mosquito feeding.
Immunohistochemical staining of RVFV nucleocapsid protein using mAb 9 as the primary antibody. Detection of viral antigen (brown) in mosquito unexposed- (A) or exposed (B) skin of the same animal. The inset in B shows staining of RVFV antigen in a macrophage. (C) Higher magnification of a blood vessel, showing margination of RVFV antigen together with neutrophils and thrombocytes with emigration of neutrophils). Notice the absence of viral staining in the cytoplasm of neutrophils (arrowheads). (D-H). positive staining (arrowheads) of endothelial cells (D), smooth muscle cells of the tunica media (E), lipocytes (F), keratinocytes (G) and fibroblasts (H). Bar = 200 μm (A, B), 100 μm (G), 50 μm (E, F) and 20 μm (C, D, H).