We evaluated the effect of holding temperature and time between mosquito death and processing mosquito pools for virus detection on our ability to detect West Nile (WN) viral RNA from pools of mosquitoes by reverse transcriptase-polymerase chain reaction (RT-PCR). Pools of 24 uninfected Culex pipiens L. mosquitoes were "spiked" with either a single Cx. pipiens that had been inoculated previously with WN virus or with an uninfected mosquito. These pools were held dry at 20, 4, -20, or -70 degrees C for selected time intervals before all mosquito pools were triturated in TRIzol LS reagent and processed for detection of WN viral RNA. While infectious virus virtually disappeared from pools maintained at 20 degrees C by 48 h after mosquito death, neither holding temperature (20 to -70 degrees C) nor holding period (up to 2 wk) affected detection of WN viral RNA by real-time RT-PCR. These findings suggest that we need not keep mosquitoes chilled to be able to detect WN viral RNA effectively by RT-PCR. This should enhance the feasibility of field-based WN virus surveillance programs where only detection of WN viral RNA is the objective and maintenance of a cold chain may not be possible.

Two recent outbreaks of locally acquired, mosquito-transmitted malaria in Virginia in 1998 and 2002 demonstrate the continued risk of endemic mosquito-transmitted malaria in heavily populated areas of the eastern United States. Increasing immigration, growth in global travel, and the presence of competent anopheline vectors throughout the eastern United States contribute to the increasing risk of malaria importation and transmission. On August 23 and 25, 2002, Plasmodium vivax malaria was diagnosed in 2 teenagers in Loudoun County, Virginia. The Centers for Disease Control and Prevention (CDC) deemed these cases to be locally acquired because of the lack of risk factors for malaria, such as international travel, blood transfusion, organ transplantation, or needle sharing. The patients lived approximately 0.5 mi apart; however, 1 patient reported numerous visits to friends who lived directly across the street from the other patient. Two Anopheles quadrimaculatus s.l. female pools collected in Loudoun County, Virginia, and 1 An. punctipennis female pool collected in Fairfax County, Virginia, tested positive for P. vivax 210 with the VecTest panel assay and enzyme-linked immunosorbent assay (ELISA). In addition, 2 An. quadrimaculatus s.l. female pools collected in Montgomery, Maryland, tested positive for P. vivax 210. The CDC confirmed these initial results with the circumsporozoite ELISA. The authors believe that this is the 1st demonstration of Plasmodium-infected mosquitoes collected in association with locally acquired human malaria in the United States since the current national malaria surveillance system began in 1957.

The Department of Defense (DoD) has engaged in West Nile virus (WNV) surveillance and response since 1999. In 2002, the three Services continued their cooperative, multidisciplinary approach to the WNV outbreak. Activities included a doubling of mosquito surveillance and vector control responses, extension of and doubling of bird and nonhuman mammal surveillance to all four continental United States regions, expanded diagnostic testing by DoD laboratories, and installation environmental clean up and personnel protection campaigns. Medical treatment facilities conducted passive surveillance and reported possible cases in DoD health care beneficiaries. Efforts were coordinated through active communication within installations, with commands, and with surrounding communities. Undertaken activities complemented each other to maximize surveillance coverage. The surveillance detected WNV on 44 DoD installations. It led directly to vector control and prevention activities, and there were no confirmed cases of WNV reported in the DoD force. This multi-Service effort is a surveillance template for future outbreaks that threaten DoD force health.

Rearrangements of 2-Bicyclo[3.2.0] heptyl Grignard Reagents ratio of 25/24 was 17/83 after 15 min; the ratio did not change after an additional 40 min at -100 "C. An additional 1.1 equiv of n-butyllithium was added. After 15 min the above ratio was 75/16; compound 26 w,ts also detected. The mixture was continually stirred at -100 "C; examination of aliquots showed that the amount of 24 decreased while the amount of butylated products (26 and 27) increased. The mixture was quenched with water after a total of 4 h after the second addition of n-CaHgLi. The mixture of products obtained contained phenylpropionitrile (25, 62%), abutyl-0-phenylpropionitrile (26, 19%), a-butyl-P-(p-butylphen-y1)propionitrile (27, 13%), and an unidentified product. Products were collected by preparative GLC.a-lButyl-j3-phenylpropionitrile (26): NMR (CDC13) 6 0.9 (t, 3, aliphatic H), 1.55 (m, 6, aliphatic H), 2.9 (m, 3, aliphatic H), 7.3 (m, 5, aromatic H).Anal. Calcd for C13H17N: C, 83.37, H, 9.15; N, 7.48. Found: C, 83.55; H, 9.05; N, 7.55. a-Butyl-8-(p-butylpheny1)propionitrile (27): NMR (CDCl3) 6 0.70-1.9 (m, 16, aliphatic H), 2.2 (m, 2, CHz), 2.85 (m, 3, aliphatic H), 7.2 (m, 4, aromatic H). Anal. Calcd for C17H25N: C, 83.89; H, 10.35. Found: C, 83.79; H, 10.60.p-Iodobenzylnitrile (28). E~a m i n a t i o n~~ of an aliquot, quenched with water taken after 15 min from reaction of 2tIz7 with 1 equiv of n-C4HgLi at -100 "C, showed the ratio of benzylnitrile to starting material (28) to be 80/20; starting material immediately disappeared upon addition of an additional 0.5 equiv of n-C4HgLi. Attempts to trap the anionic products from the reaction mixture with cyclohexanone gave a multicomponent mixture (GLC) which was not resolved. (24) Shown by coinjection of an authentic sample prepared from benzyl nitrile (1 equiv), sodium hydride (2 equiv), and methyl iodide (2 equiv) in dirnethylformamide: bp 226 OC (754 mm); n Z 5~ 1.5016 (lit.25 bp 232The Grignard reagent (12) from 2-bromobicyclo[3.2.0]heptane undergoes a ring cleavage rearrangement to cyclopentenylethyl (13) and cycloheptenyl (14) Grignard reagents. Grignard 14 is slowly converted to 13. The rate of rearrangement of 12 is thought to be somewhat retarded by geometric restrictions introduced by the bicyclic skeleton. The facility of the rearrangement of 12 4 14 may indicate that the preferred transition state for rearrangement is nonplanar. Rearrangement of the Grignard reagent 25 from 3-chlorotricyclo[5.3.0.02~6]decane occurs in analogous fashion. Grignard reagents 31 from 2-bromo-6-alkoxybicyclo[3.2.0]heptanes decompose with elimination of the alkoxy group and ring cleavage to 3-vinylcyclopentene and 1,4-cycloheptadiene.

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