2.10 Goat virus inoculations and mosquito challenge
Mature female, non-pregnant dairy goats of multiple breeds were acquired
from a commercial dairy and housed in an Animal Bio-Safety Level 3
facility for the duration of the experiment. Goats were inoculated with
5.6 log10 PFU freshly grown MP-12 or 6.6
log10 PFU DDVax, as determined by plaque assay. Blood
was drawn from goat jugular vein at days 1, 2, and 3 post-inoculation
into gel serum separator tubes (Becton Dickson, https://www.bd.com/);
serum was collected by spinning at 1200 x g for 10 minutes. Serum
was aliquoted and stored at -80oC. Serum samples were
titered by plaque assay, and RNA was extracted for detection and
quantification of viral RNA.
For mosquito feeding, goats were manually restrained, and mosquitoes
were held in cartons with mesh bottoms against patches of clipped fur
and held for about 30 minutes to allow feeding on days 1 and 2
post-inoculation (Figure S1). Since Cx. tarsalis mosquitoes did
not feed well on goats, on day 3 post- inoculated, mosquitoes were
exposed in the laboratory to freshly-collected goat blood (collected
into EDTA tubes (Becton Dickson,https://www.bd.com/)) using a water
jacketed feeding apparatus heated to 37oC. Engorged
mosquitoes were held for 7 days at 28oC, rH 80%. At 7
days-post-feeding, bodies and legs/wings were placed in individual tubes
containing mosquito diluent (see above). Samples were homogenized on a
Qiagen Tissuelyzer (Qiagen) at 30 beats per second frequency for 30
seconds, then pelleted at 14,000 x g in a microfuge at
4oC for 3 minutes. Tubes were stored in
-80oC. Infectious virus (CPE+/-) was measured by
plaque assay using 100 µl undiluted sample in duplicate to determine the
frequency of mosquito bodies bearing infectious DDVax virus or MP12 RVFV
(control). For those with RVFV-positive bodies, legs/wings were also
titrated by plaque assay to determine the frequency of mosquitoes with
disseminated infectious virus.