Vector Competence Of Aedes Albopictus Infected With Zika And Dengue Virus

Background:Zika virus(ZIKV)and dengue virus(Dengve virus,DENV)are mosquito-borne infectious disease which became pandemic last years.The ZIKV outbreak in French Polynesia from 2012 to 2014 shocked the public health communities because of the high prevalence Guillain Barre Syndrome(GBS)cases.Worse than that,the ongoing ZIKV epidemic in the Americas since 2015 was associated with congenital infection and unprecedented number of microcephaly in newborn infants.Dengue virus infection in humans was often inapparent but can lead to a wide range of clinical manifestations,from mild fever to potentially fatal dengue hemorrhagic fever and dengue shock syndrome.Considering the imported cases of ZIKV had been reported in China,what are the potential vectors for ZIKV in China?In addition,to evaluate the effect of temperature on the vector competence of Aedes albopictus for DENV-2 will provide a guide for vector control and prevention of mosquito-borne diseases.Objective:Our goal is to establish a model of cells and mosquitoes infected with ZIKV and DENV.We identified the vector competence of Aedes aegypti,Aedes albopictus and Culex quinquefasciatus for ZIKV,in China.C6/36 cell and Aedes albopictus were used for infection with DENV-2.We compared the differences of the vector competence of Aedes albopictus for DENV-2 under different temperature condition.Methods:Vero,C6/36,Aag2 cells and suckling mouse were infected with ZIKV.and the amount of ZIKV was detected by qRT-PCR.The location of ZIKV particles in C6/36 cells was determined by indirect immunofluorescence(IFA).The collection method of ZIKV was ascertained by the infection status of Ae.albopictus orally infected with blood meal which was prepared by mixing defibrinated sheep blood with the whole cell suspension,the supernatant or the cell supernatant after freeze thawing at a ratio of 1:2 supernatant.Ae.albopictus was infected according to the best conditions of ZIKV proliferation and collection.The infection rate was tested by RT-PCR and the ZIKV titre was determined by qRT-PCR.After the infection model and the detection system was established,Ae.aegypti,Ae.albopictus and Cx.quinquefasciatus were starved for 24-48 h and exposed to the infectious blood meal for 30 min.Fully engorged females were transferred to HP400GS incubator(28°C,80%relative humidity and light/dark cycle of 16h:8h).ZIKV infection rate and viral genome of the whole mosquitoes were detected by RT-PCR and qRT-PCR at 0,4,7,10 and 14 days post infection(dpi).Another batch of Ae.aegypti,Ae.albopictus and Cx.quinquefasciatus were infected by ZIKV.The migdut,head,salivary glands and ovaries of each mosquito were dissected at the above mentioned point in time.The infection status from each tissue was detected by RT-PCR and the viral RNA copies from the positive samples were quantified by qRT-PCR.The cytopathic effect was observed when C6/36 cells were infected with DENV-2.DENV-2 in C6/36 cells was assayed by RT-PCR.The DENV-2 titre was determined by TCID50 and the presence of DENV-2 partiles in cells was observed by IFA.Ae.albopictus was orally infected with the fresh viral supernatant mixing with defibrinated sheep blood and the mosquitoes infection rate was detected by RT-PCR.Ae.albopictus infected with DENV-2 was placed at 18℃,23℃,28℃ and 32℃,respectively.The midgut,ovaries and salivary glands of each mosquito were dissected at 0,5,10 and 15 dpi.The infection status from each tissue was detected by RT-PCR and the viral RNA copies from the positive samples were quantified by qRT-PCR.Immunohistochemical was used to observe the viral antigen of midgut and salivary glands at mosquito placed at 32℃ for 15 days.Results:Compared to Aag2,Vero cells and suckling mice,the virus titer was the highest when C6/36 cells infected with ZIKV were placed at 28℃ for 5 days.The whole cell suspension was the best viral collection method,which lead to the high mosquito infection rate.The positive rate of ZIKV in Ae.albopictus 27.27%～30.77%and the virus titer was gradually increased along with the prolongation of infection time.Ae.aegypti and Ae.albopictus maintained a high infection rate at all time points and the virus titer was increased gradually.A small amount of infection rate and low Titre of ZIKV at 4 dpi and 7 dpi were detected in Cx.quinquefasciatus.No ZIKV was detected from the whole mosquito and midgut of Cx.quinquefascitus after 7 dpi.The ZIKV in the heads and salivary glands of Ae.aegypti started from 4 dpi.However,the spread of ZIKV in the heads and salivary glands of Ae.albopictus was detected at 7 dpi and 10 dpi,respectively.Cytopathic effect was very obvious when C6/36 cells infected with DENV-2 at 37℃ for 2 d.The specific DENV-2 fragment was amplified by RT-PCR.The DENV-2 titre detected by TCID50 was 107.625/mL.IFA confirmed DENV-2 particles in C6/36 cells.Ae.albopictus showed a different level of mosquito infection rate with DENV-2.Then Ae.albopictus was orally infected with DENV-2 and reared at different temperature.18℃,DENV-2 was slowly proliferation only in the midgut of Ae.albopictus and the virus could not spread to the salivary glands.Between 18℃ and 32℃,following the increased temperature,the replication of viruses in mosquitoes was accelerated,and the incubation period was decreased.DENV-2 detected in the ovaries and salivary glands was 10 dpi when Ae.albopictus was maintained at 23℃ and 28℃.The infection rate and viral titre of 28℃ was higher than that of 23℃ at all time point.When Ae.albopictus was maintained at 32℃,DENV-2 detected in the ovaries and salivary glands was only 5 dpi.The virus antigen in the midgut and salivary glands could be observed by immunohistochemistry.Conclusion:C6/36 cell is an ideal model for proliferation of ZIKV and DENV-2.Ae.albopictus was successfully infected by ZIKV and DENV-2,which was confirmed by RT-PCR and qRT-PCR.China’s Ae.aegypti and Ae.albopictus are susceptible whereas Cx.quinquefasciatus is not able to transmit the imported ZIKV.Comparatively,the vector competence of Ae.albopictus is less superior to Ae.aegypti,but considering the widely distribution,Ae.albopictus might become the primary vector for ZIKV in China.Temperature is an important factor affecting the vector competence of Aedes albopictus for DENV-2.During the suitable temperature range(18℃-32℃),DENV-2 dissemination and transmission can be observed more quickly when the temperature was higher because of the faster proliferated virus.