| Background:Zika virus(ZIKV),a mosquito-borne flavivirus,is known around the world because of its associated serious neurological complications,such as Guillain-Barre syndrome in adults and microcephaly in newborns,which pose serious threats and have become a public health emergency of international concern.It had been reported that after ZIKV infection during pregnancy,the virus replicates and crosses the placental barrier.Vertical transmission is the main route of ZIKV-induced microcephaly in the fetus.Many flaviviruses,including dengue,yellow fever,West Nile,and St.Louis encephalitis virus,can be transmitted vertically from infected female mosquitoes in the Aedes genus to their offspring.Evidence of ZIKV infection in field-collected eggs and mosquitoes has been reported in different countries.ZIKV-positive offspring have been reported in the literature,with different filial infection rates,while negative results in F1 offspring have also been reported.In Ae.aegypti,the filial infection rate ranged from 0 to 6.99%,in Culex quinquefasciatus,the filial infection was between 0.04%and 1.52%,while the filial infection rate in Ae.albopictus ranged from 0 to 1.18%.These varied and even contradictory reports may be due to different experimental designs,such as the intrathoracic inoculation of ZIKV or the testing of pooled larvae rather than the assessment of individual mosquitoes after ingestion of an infectious blood meal.At present,the characteristics of vertical transmission of ZIKV in vector mosquitoes are not yet completely known.In addition,microcephaly is caused by the vertical transmission of ZIKV from an infected mother to her infant.Does the vertical transmission of ZIKV cause a similar pathological effects in mosquito hosts?Objective:Aedes albopictus is an important vector of ZIKV globally and a potential primary vector in China.To date,most experiments have focused on the vertical transmission of ZIKV in Ae.aegypti,while studies on another susceptible mosquito,Ae.albopictus,are very limited.To explore the questions above,a series of laboratory studies were carried out to characterize the vertical transmission of ZIKV in Ae.albopictus.Methods:Female Ae.albopictus were starved for 24-48 h and were infected by allowing them to feed on an infectious blood meal containing 0.8 ml of ZIKV and 0.4 ml of defibrinated sheep blood.The midguts and ovaries of 10 mosquitoes were dissected during the first gonotrophic cycle(FGC),corresponding to 4,7,10,and 14 dpi.The tissues were cold homogenized separately.Total RNA extraction,primer design,ZIKV cDNA synthesis,amplification of the NS1 region of ZIKV by reverse transcription PCR(RT-PCR)and the quantification of ZIKV RNA copy numbers were performed according to standard procedures.For those mosquitoes with ZIKV-negative midguts,we did not further analyze the ovaries.The ovaries of positive mosquitoes were dissected over the course of two gonotrophic cycles.After the FGC,the remaining mosquitoes were transferred into microcosms with moist filter paper for oviposition.Then,the mosquitoes were fed with pure sheep blood after starvation for 24 h.The ovaries were dissected and ZIKV was detected at during second gonotrophic cycle(SGC),corresponding to 23,26,29,and 33 dpi.The experiment was carried out with 240 samples in total.In the immunohistochemistry assay(IHC),after removal of the legs and wings,the infected mosquitoes were harvested at 14 dpi.The entire body was embedded in paraffin and serially cut for histologic examination.The slides were stained with mouse monoclonal to ZIKV NS1 protein antibody and horseradish peroxidase(HRP)-conjugated anti-mouse IgG secondary antibody.Following the manufacturer's protocol,3,3'-diaminobenzidine and hematoxylin were applied,and the specimens were examined under a light microscope.Tissues dissected from mosquitoes fed on an uninfected blood meal were used as the control.In the vertical transmission assay,starved female mosquitoes were separated into four groups(groups ?,?,?,and ?,42 mosquitoes in each group).Individual mosquitoes from group ? were transferred to a paper cup with filter paper after being fed an infectious blood meal.The mosquitoes laid eggs from 4 to 7 dpi,and the eggs were collected.After counting,the eggs on the single filter paper were placed into a tube,considered one pool,for detecting ZIKV.Immediately after,the mosquito was starved for 24 h and refed with pure sheep blood to stimulate oviposition.These eggs were collected in a new tubes.The ovaries of the surviving mosquitoes were dissected,and only the surviving mosquitoes with ZIKV-positive ovaries who laid eggs twice were studied.Mosquitoes from group ? were fed only a sheep blood-C6/36 cell mixture as a control,whereas those in group ? were provided with a sheep blood-ZIKV mixture as a treatment.The ovaries of the experimental groups were dissected directly after the first oviposition.The eggs of these two groups were reared to adulthood.The male and female progeny in the treatment groups were dissected as follows:males:testis and accessory glands;females:salivary glands.The tissues were cold homogenized separately for detecting ZIKV.The control groups were also examined to compare the hatching,pupation,and emergence rates.Mosquitoes from group IV were transferred to microcosms for oviposition.The eggs were collected and reared to adulthood.Thirty female offspring were allowed to feed on one C57BL/6 mouse(one day old).Mouse blood was collected to detect ZIKV at 7 dpi.Blood from each mouse was collected again before the suckling mice were euthanized at 14 dpi.The experiment was carried out with 13 mice and 390 female mosquito offspring in total.Results:A total of 240 Ae.albopictus females were used to measure the infection rate in the ovaries.RT-PCR and real-time reverse transcription PCR(RT-qPCR)were used to detect the ZIKV quality and quantity,and IHC was carried out to locate the virus protein in the tissues.At 0 dpi,the infection rate in the midgut was 100%.Staring from 4 dpi,the ovaries showed infection evidence and remained positive throughout the remainder of the experiment.The average infection rate of the ovaries in the SGC(93.75%)was significantly higher than that in the FGC(40.66%).The average virus titer in the ovaries in the SGC(6.10±2.60)was higher than that in the FGC(4.90±2.46).In addition to quantitative detection by RT-qPCR,ZIKV antigen was detected in the germarium of the ovary and the embryo by IHC.No pathogenetic injury was found in the ZIKV-positive ovaries compared with the control ovaries.When the ovaries were dissected at 14 and 33 dpi,the number of eggs in the ovaries was counted to evaluate ovarian development.Without a mature embryo in the ovary would be regarded as undeveloped.To evaluate oviposition,another batch of ZIKV infection mosquitoes was allowed to lay eggs at 14 and 33 dpi.Ovaries were dissected to count the remaining eggs.The control group of the mosquitoes were fed with an uninfected blood meal.The development rates of infected ovaries at 14 and 33 dpi did not show any significant differences from that in uninfected mosquitoes.At 14 dpi,the number of eggs in the ZIKV group was similar to that in the control group.However,at 33 dpi of the SGC,the number of eggs in the control groups was significantly higher than that in the ZIKV groups.Oviposition rates were calculated,and no significant difference was detected.In the vertical transmission experiment,the infection rate in the ovaries was 100%.Regarding the egg pools,the infection rate in the FGC(47.62%)was not different from that in the SGC(33.33%).The amount of ZIKV in the egg pools was calculated,and the log 10 levels in the FGC(4.48±1.89)were not different from those in the SGC(4.25±1.33).In the control group,the rates of hatching,pupation and emergence were 61.01%,93.08%and 95.15%,respectively,while the rates in the ZIKV group were 57.49%,91.19%and 98.12%,respectively.No significant difference was found between them.In the adult F1 pool,the infection rates of males and females were 11.90%and 9.52%,respectively,and no obvious difference was found.The titer of ZIKV was 3.04±0.71 in male gonads and 2.85±0.43 in female salivary glands,suggesting that no sex tendency occurred.To evaluate the efficiency of vertical transmission of ZIKV in Ae.albopictus,filial infection rates were calculated.The minimal filial infection rates in eggs from two gonotrophic cycles were 2.06%and 0.69%,without statistical significance.The effective population transmission rate(minimum of 1.87%)was calculated in F1 adult tissues instead of F1 adult whole bodies.To evaluate the vector capacity of the offspring infected by vertical transmission,thirteen C57BL/6 suckling mice were fed on by ZIKV-positive F1 females.At 7 dpi,ZIKV was detected in two of the thirteen mice;the ZIKV titers(log10)were 2.8 and 3.6,respectively.After another 7 days after feeding,the virus titer of one infected mouse increased and reached 4.7,while the other decreased to 3.0.Conclusion:ZIKV can be vertically transmitted in Ae.albopictus via transovarial transmission.The vertical transmission rates in F1 eggs and adults were 2.06%and 1.87%,respectively.Even though the vertical transmission rates were low,the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to immunocompetent mice through feeding.The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases. |
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