Table 5.

Effects of freezing and thawing infected mosquitoes on detection of DENV in mosquito pools by virus isolation, RNA detection, and NS1 antigen detection^*

No. of freeze-thaw cycles	Pool size	Titer (log10 PFU/mL) by day PI					RT-PCR by day PI					Nested PCR by day PI					Mean A450 by day PI
		Uninfected	1	7	14	21	Uninfected	1	7	14	21	Uninfected	1	7	14	21	Uninfected	1	7	14	21
1	1	neg	1.94	4.49	3.64	3.64	neg	pos	pos	pos	pos	neg	pos	pos	pos	neg	neg	6.75	6.65	6.63	6.54
1	10	neg	1.00	4.25	3.64	3.11	neg	neg	pos	pos	pos	neg	neg	pos	neg	pos	neg	6.66	6.65	6.60	6.51
1	100	neg	0	0	0	0	neg	pos	pos	pos	pos	neg	pos	pos	neg	pos	neg	6.78	6.57	6.58	6.40
5	1	neg	1.94	4.41	3.64	2.94	neg	pos	pos	neg	pos	neg	pos	pos	pos	pos	neg	4.60	6.64	6.63	6.56
5	10	neg	1.64	4.11	3.64	0	neg	pos	pos	pos	pos	neg	pos	pos	pos	pos	neg	6.38	6.55	6.65	6.55
5	100	neg	0	0	0	0	neg	pos	pos	pos	pos	neg	pos	pos	pos	pos	neg	4.94	6.57	6.60	6.56

^*Mosquitoes at 1, 7, 14, or 21 days post-DENV infection were subjected to 1 or 5 freeze-thaw cycles and pools of 1 infected mosquito in the total pool size shown were constructed and assayed by virus titration, RT-PCR/nested PCR or NS1 Ag detection for DENV infection.

DENV = dengue virus; PI = post-infection; PFU = plaque-forming units; RT-PCR = reverse transcription-polymerase chain reaction; pos = positive, neg = negative.