P. monodon (~20 g body weight, 3 months old, n = 24) were individually
released into tanks containing 10 L of seawater at ~28 °C, 16 of
which were injected with 100 μl YHV inoculum (approximately 106
viral copies) and 8 were injected with 100 μl PBS (control). The shrimps
were fed and monitored daily. The shrimps injected with YHV displayed
severe symptoms of the disease within 5 d. From the 16 shrimps, 8 were
sampled individually 12 h after they started showing any signs of
morbidity, and an additional set of 8 specimens were sampled following
the same protocol 24 h after morbidity onset. The shrimps were rinsed
with sterilized seawater, and 100 mg of gill tissue was collected, homogenized
in 9 volumes of sterilized PBS and centrifuged at 10,000 ×g for
10 min at 4 °C. The clarified homogenate from each gill sample was
diluted 1:100 in PBS. The gills from PBS-injected shrimps were sampled
1 week after injection and processed following the same procedure. An
aliquot of each diluted gill supernatant was processed for PCR as
described