Vector competence of Aedes albopictus and Ae. aegypti (Diptera: Culicidae) to dengue 1 virus on Taiwan: development of the virus in orally and parenterally infected mosquitoes.

The vector competence in Ae. aegypti (L.) and Ae. albopictus (Skuse) from southern Taiwan to the dengue 1 virus was studied to elucidate the distribution of dengue infection during the 1987-1988 outbreak. The brain of Ae. aegypti was infected as early as 3 d after intrathoracic inoculation. The esophagus and the proboscis (tissues within the labium) were infected 5 d after inoculation. The salivary gland was highly susceptible to the virus, but no specific infection site was found. Gangli, muscles, and diverticula within the thorax were not infected. In the abdominal area, the ventral diverticula, Malpighian tubules, ganglia, and the dorsal vessel were not infected. However, the entire gut was susceptible to dengue 1 virus, although it was not infected simultaneously. Only a certain type of midgut epithelial cells was infected by the virus. The ovarioles, oviducts, and accessory glands frequently were infected. However, the spermathecae were not infected, perhaps because of the chitin-rich outer layer. Infections of the testes, vas deferens, seminal vesicles, and accessory glands of males also were detected in this study. The tissues of the proboscis were never infected in Ae. albopictus but frequently were infected in Ae. aegypti, indicating that the virus may escape the salivary gland barrier more efficiently in Ae. aegypti than in Ae. albopictus. When these mosquitoes were fed on hanging drops, the salivary gland infection and transmission rates of Ae. aegypti were always higher than those of Ae. albopictus. On Taiwan, Ae. aegypti appears to be a more competent vector in the transmission of the dengue 1 virus than Ae. albopictus.

Induction of cytochrome P-450 activities by nicotine in the tobacco hornworm,Manduca sexta.

The induction by dietary nicotine of a series of cytochrome P-450 enzyme activities was investigated in early fifth-instarManduca sexta larvae. At a low nicotine concentration in the diet (0.1 %), three of 12 midgut microsomal enzyme activities were significantly increased. At a higher concentration (0.75%) commonly found in plants of the genusNicotiana, nine of 12 activities were induced by 1.4- to 10.0-fold. Total cytochrome P-450, P-450 reductase activity, and midgut microsomal metabolism of nicotine were also increased by feeding 0.75% nicotine. Nicotine was metabolized by midgut microsomes to nicotine-1-N-oxide and cotinine-N-oxide. Fat body microsomal nicotine metabolism was low and unaffected by dietary nicotine. Isolated nerve cords were able to metabolize nicotine in vitro but this metabolism was not inducible by dietary nicotine. Nicotine-fed fifth-instarM. sexta larvae showed an increased tolerance to subsequent nicotine injection when compared to larvae fed a control diet. These results support the idea that induction of midgut cytochrome P-450-related metabolism is an adaptation ofManduca sexta to dietary nicotine.

Identification of a very early promoter of insect Hz-1 virus using a novel dual-expression shuttle vector.

Very early promoters of viruses control the proper cascade expression of viral genes and are essential for completion of virus life cycles. These promoters are usually rare and weak and do not encode structural proteins. As a result, they are difficult to identify. In order to identify and clone the very early promoters of a large eukaryotic DNA virus, the Hz-1 virus, a novel cloning strategy was applied. This strategy is based on a dual-expression shuttle vector containing a promoter-less lacZ gene. Insertion of eukaryotic promoters upstream permits the efficient expression of LacZ in bacteria cells. The function of the putative promoters was then confirmed by their proper expression in insect cells. The first two productive infection-specific promoters of Hz-1 virus, contained within the shuttle vectors pTSV-2-129 and pTSV-2-49, were cloned from the HindIII-K and HindIII-A fragments of the Hz-1 viral genome, respectively. By primer extension analysis, an immediate and constitutive expression of the promoter in clone pTSV-2-129 was detected after viral infection. Identification of the productive infection-specific promoters has laid down important groundwork for future studies on the molecular mechanism of the transcriptional switch between productive and persistent infections of Hz-1 virus.

Effects of calcium channel antagonists on the corpora allata of adult male loreyi leafworm Mythimna loreyi: juvenile hormone acids release and intracellular calcium level.

The effects of voltage-dependent calcium channel (VDCC) antagonists and the non-specific calcium channel antagonists on both juvenile hormone acids (JHA) release and cytosolic free calcium concentration ([Ca2+]i) are investigated in the corpora allata (CA) of the adult males loreyi leafworm Mythimna loreyi. The VDCC antagonists used in this study are: the L-type antagonists diltiazem, nifedipine, and verapamil, the N-type antagonist omega-Conotoxin (CgTx) GVIA, the N- and P/Q-type antagonist omega-CgTx MVIIC, and the T-type antagonist amiloride. The non-specific calcium channel antagonists used in this study were cadmium (Cd2+), cobalt (Co2+), nickle (Ni2+), and lanthanum (La3+). The results show that both the DHPs-sensitive L-type antagonist nifedipine and the N-type antagonist omega-CgTx GVIA were able to inhibit JHA release, but only omega-CgTx GVIA was able to reduce the [Ca2+]i. Among the non-specific calcium channel antagonists, Cd2+ is the most potent in reducing JHA release but without obvious effect on the [Ca2+]i, La3+ significantly increases the [Ca2+]i but without effect on JHA release.

Sensitization to Blomia tropicalis among asthmatic patients in Taiwan.

House dust mites have been reported as one of the most important allergens in Taiwan especially in asthmatic patients. This study was conducted to determine the allergenicity of Blomia tropicalis and sensitization of asthmatic patients in Taiwan. Serial dust samples were collected every month between July 1993 and June 1994 from 13 houses of mite-allergic patients. About 1 m2 surface area of a quilt was vacuumed. The floating method was used to collect mites, then identification and counting were performed. Results showed that Dermatophagoides pteronyssinus and B. tropicalis were the two most common species of mites found in allergenic patients' houses in Taipei. D. pteronyssinus accounted for 52.1% of the total number of mites and was found in every house. B. tropicalis, although not present in every sample, accounted for 44.3% of the total number of mites. The skin test positive reaction to B. tropicalis, D. pteronyssinus and Dermatophagoides farinae were 73.3, 88.3 and 85.0% as determined from 60 allergic patients who attended our allergy clinics. The extract prepared from B. tropicalis was used to determine the allergenicity and contained at least 30 protein components when silver stained. The most frequently detected allergens were proteins with molecular weights of 14.3, 106.5, 94.0, 72.0, 91.9, 63.7, 100.3, 43.6, 27.3, 62.0, 34.7, 18.3, 41.1 and 21.9 kD. The frequency of IgE binding of patient sera to those proteins were 87.0, 65.2, 56.5, 43.4, 39.1, 39.1, 34.8, 30.4, 30.4, 17.4, 17.4, 17.4, 13.0 and 8.7%. The results from immunoblot inhibition showed that there was IgE cross-reactivity among the B. tropicalis and D. pteronyssinus. However, there were two major allergenic components of B. tropicalis not inhibited by D. pteronyssinus with molecular weights of about 14.3 and 27.3 kD. The use of B. tropicalis extract for diagnostic purposes to identify patients with specific sensitivity should be considered in Taiwan.

A 2.9-kilobase noncoding nuclear RNA functions in the establishment of persistent Hz-1 viral infection.

Differential viral gene expression during both productive and persistent infections of Hz-1 virus in insect cells was elucidated. Despite more than 100 viral transcripts being expressed during productive viral infection, massive viral gene shutoff was observed during viral persistency, leaving the 2.9-kb persistence-associated transcript 1 (PAT1) as the only detectable viral RNA. Persistence-associated gene 1 (pag1), which encodes PAT1, was cloned and found to contain no significant open reading frames. PAT1 is not associated with the cellular translation machinery and is located exclusively in the nucleus. Further experiments showed that PAT1 is functional in the establishment of persistent Hz-1 viral infection in the cells. All the evidence collectively indicates that PAT1 is a novel nuclear transcript of viral origin. Our results showed that although PAT1 and XIST RNA, a mammalian X-inactive specific transcript, are transcribed by different genes, they have interesting similarities.