The Pathogenicity Preliminary Study And Establishment Of Real-time Fluorescent PCR Detection Method Of NDiV

ObjectiveNam Dinh virus(hereinafter referred to as NDV) is a new arbovirus which was discovered recently. It was the first time that in the country our group isolated it from the adult mosquitoes successfully, but the virus detecting methods and epidemiological significance is still rarely reported. This study was designed to establish a real-time PCR detection method which can specific and rapid detect the adult mosquitoes with NDiV, providing a reference to rapid detect the mosquitoes carrying NDiV in the environment. Through animal experiments to understand the NDiV pathogenic role, providing the basis for the direction and control of vector-borne infectious diseases. MethodsCulturing Nam Dinh virus with C6/36 cells and centrifugal purified virus after 4-6 days when the cells appear obvious cytopathic, using 50% cell end-point method to evaluate the virus titers. The titer of NDiV which were already isolated were cultured in mosquito cells(C6 / 36 cells) and mammalian cell, then observing the growth status of NDiV grow in different cells. Animal experiments:(a) After the grouping processing of 3-4-day-old suckling mice, then the isolated virus was inoculated with different doses in neonatal rat brain, abdominal and nasal. Observing the incidence of the suckling mice and making a record as well as extracting the incidence of tissue’s nucleic acid slurry to do RT-PCR detect and virus identification.(b) After the grouping processing of 6-day-old SPF chick embryo then inoculating different doses of virus which were isolated in SPF chick embryo yolk sac, allantois, amniotic and observing the incubated situation.(C) After the grouping processing of chicks, by intracerebral inoculation, wings inoculation and oral ways to inoculate virus which was isolated. Then mark the numbers of the chicks which appear lesions. Collecting suckling mice’s and chick embryos’ diseased parts or tissues, extracting nucleic acid after grinding to do RT-PCR detection. The above experiments are set the control groups.To compare the sequence alignment of Vietnamese NCBI and China’s NDiV virus, selecting RdRp gene in conserved region and designing specific primers and TaqMan-MGB probe. By optimizing the method in specificity, sensitivity, stability and applicability which was established by experiments to establish a high specificity, strong sensitivity real-time PCR detection method. ResultsDetecting the growth of NDiV isolates in mosquito cells(C6 / 36 cells) and mammalian cell by RT-PCR detection. The test results showed that the virus only proliferated in mosquito cells but not proliferated in mammalian cells. NDiV had significantly cytopathic effect on C6 / 36 cells and C6/36 cells are sensitive to NDiV. By C6/36 cells in 50% of the end-point method that calculated NDi V virus titer is: 1.41×106 PFU /ml. After different parts of suckling mice and chick embryos were inoculated different doses of NDiV, there was no significant Pathogenesis.By several times optimizing test that at the first time, a detecting method NDiV MGB probes for RT-PCR was established. This method of NDiV is short time-consuming and high sensitivity, and the low detection limit is 1PFU. The method has good specificity which has no cross-reaction with dengue virus serotypes 1-4, Japanese encephalitis virus, rotavirus, respiratory syncytial virus, adenovirus, astrovirus. Four different nucleic acid content of NDiV standard was tested for five times, the average coefficient of variation range of Ct was 1.67%-3.68%, and thus it was high stability. Through testing, Longgang District the mosquitoes carrying NDi V was 18%, and the residential areas, hospitals and stadiums have highest proportion of mosquitoes carrying NDiV, which were 22%, 25%, 31%. Mosquitoes carrying NDi V were lower proportion in other area. ConclusionsC6/36 cells are sensitive cells which are susceptible to be infected by NDiV. NDiV can cause Lesions effects in C6/36 cells but it can’t grow and copy in the mammalian cells. According to experimental observation, NDiV may not have pathogenic effect on suckling mice and chick embryos. NDiV of TaqMan-MGB probe RT-PCR method is a specific, rapid, high sensitivity and good stability method which can be applied to NDiV epidemiological surveillance, improving the capacity for rapid detection of the virus.