| Dengue fever/dengue haemorrhagic fever (DF/DHF) is one of the most important human arbovirus infectious diseases in tropical and subtropical areas. Dengue (DEN) virus is an enveloped arbovirus with a positive single stranded RNA genome and transmitted to man by the bite of a domestic mosquito, Aedes aegypti being the principal vector although some other species such as Ae. albopictus is of importance. The epidemiology, especially the primitive enzootic transmission cycle of DEN virus is more important. A series of investigations has confirmed that dengue virus can be transmitted transovarially by mosquitoes in nature, and the diffusion of Ae. albopictus mainly depends on diapausing eggs. It is important to explore whether the DEN-2 can survive in diapausing eggs and diffuse to all over the world by human activity such as tire trade etc. The present studies focused on the item and the results reported below.Mosquitoes of Guangzhou strain of Ae. albopictus were used and they were infected orally with dengue 2 virus. Eggs from 3 gonotrophic cycles were collected, one of the groups were subjected to a simulated overwintering condition with a low temperature, short photoperiod and desiccate environment conditions (4C, RH=75%, L/D=8/16) to induce into diapausing situation, a second group of eggs were transferred to normal laboratory condition with desiccation (25C, RH=75%, L/D= 14/10). First, both semi-nested PCR and virus isolation with C6/36 cell were used for assay the DEN-2 in eggs. Furthermore, nucleic acid hybridization was used for the detection of virus RNAs, to confirm whether DEN-2 can survive in diapausing eggs and continue to replicate. After terminating diapause by artificial inducing, a further study was devoted to determine whether DEN-2 virus can be transmitted transovarially and horizontally by detecting virus in F| progeny mosquitoes. In addition, hatching attempts of diapausing eggs and productive capacity of F, progeny adult were also observed in order to explore the maintenance mechanism of arbovirus in nature and the relationship between vectors, virus and host. The results are asfollows.1. Detection and isolation of DEN-2 in diapausing eggs and normal preserved eggs of infected Ae. albopictusSemi-nested PCR screening was used to detect DEN-2 in diapausing and normal preserved eggs from infected Ae. albopictus, the results were confirmed in the later with virus isolation in C6/36 cells from those eggs. It appears that semi-nested PCR amplication might be reliable to detect DEN-2 in the two kinds of eggs.2. The replication of DEN-2 in eggs and its detection methodsSingle strand DNA product from the asymmetric PCR was labeled with DIG as a pair of probes, antisense probe and sense probe to detect viral RNA. Of the two probes, antisense probe was used to detect sense RNA of DEN-2 virus, and the other to detect antisense RNA. Meanwhile, the sense probe was applied specifically to detect and distinguish RI RNA from genomic RNA precipitated by 4mol/L LiCl solution, in which RF RNA were suspended supernatant. The antisense probe was used to identify RF RNA by slot-blot analysis.The sense RNA and antisense RNA of DEN-2 were detected in eggs both in diapausing and normal preserved condition by southern blot analysis with antisense probe and sense probe, respectively. Although the level of viral antisense RNA in diapausing eggs is very low, it indicates that few antisense RNA of DEN-2 was still synthesized in diapausing eggs. And then, RI and RF RNAs were also detected in normal preserved eggs. However, neither RI RNA nor RF RNA was detected in diapausing eggs and few viral antisense RNA was detected by southern blotting. In diapausing eggs, viral RNA activites were stopped in the period of antisense RNA to RI RNA. Compared with those in normal preserved eggs, the virus in diapausing eggs seemed to be inactive. Factually, during the period the cultivation of DEN-2 with C6/36 cells, it was found that cytopathic effect(CPE)occurred 12h later in C6/36 cells with DEN-2 in diapausing eg... |
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