Experimental Zika virus infection in Aedes aegypti: Susceptibility, transmission & co-infection with dengue & chikungunya viruses.

BACKGROUND & OBJECTIVES: There are reports about the susceptibility of Aedes mosquitoes to ZIKV from various countries, however, no such information is available from Indian sub-continent, although, high level of group cross-reactivity of ZIKV with other flaviviruses has been reported. During outbreak situations, many cases of Dengue (DEN) and Chikungunya (CHIK) are reported. In such scenario, vector mosquitoes are likely to get co-infection/secondary-infection with one or other virus. The present study was carried out to determine the susceptibility of Indian strain of Aedes aegypti to Zika virus (ZIKV) strain (MR-766) and the effect of co-infection/super-infection with either dengue virus (serotype-2) (DENV) or chikungunya virus (CHIKV) on ZIKV replication. METHODS: Ae. aegypti mosquitoes used in this study were reared for many generations since 1980 at laboratory colony maintained at the ICMR-National Institute of Virology, Pune, India. Transmissibility of ZIKV from infected mosquitoes to suckling mice was also studied. Mosquitoes were experimentally infected with ZIKV and super-infected with either DENV or CHIKV via membrane-feeding route and incubated for 14 days at 28±2°C and humidity of 85±5 per cent. Replication of these viruses in mosquitoes was confirmed using real-time reverse transcription-polymerase chain reaction and immunofluorescence assay. Twenty infected mosquitoes were allowed to feed upon four suckling CD1 mice for about 30 min. Transmission of the ZIKV by infected mosquitoes to suckling mice was confirmed by the appearance of clinical signs and the presence of viral RNA in different organs. RESULTS: Concomitant infection of mosquitoes with all the three viruses showed simultaneous propagation of all three viruses, confirmed by real time RT-PCR and IFA. Infection of mosquitoes with CHIKV followed by ZIKV showed positivity in individual head squashes (7%) for both viruses using IFA; only 8.3 per cent showed dual positivity with primary infection of ZIKV followed by DENV; 8.3 per cent dual infection positivity was observed when infected with DENV followed by ZIKV; 5 per cent showed dual infection was observed when infected with ZIKV followed by CHIKV. Ae. aegypti was found to be susceptible to ZIKV strain as ZIKV could be detected from the second post-infection day (PID) in infected mosquitoes. Transmission of ZIKV to mice by the bite of infected Ae. aegypti establishes this species as a potential vector. INTERPRETATION & CONCLUSIONS: From super-infection experiments, it was concluded that ZIKV might have a relative advantage in replication dynamics over DENV. Vertical transmission was not observed for ZIKV in experimentally infected mosquitoes (n=920 larvae). Further studies are required to understand the possibility of silently circulating ZIKV in India, which remain non-detected because of lack of surveillance.

Persistence of viral RNA in chikungunya virus-infected Aedes aegypti (Diptera: Culicidae) mosquitoes after prolonged storage at 28°C.

Experiments were conducted to determine the persistence of chikungunya viral (CHIKV) RNA in experimentally infected Aedes aegypti mosquitoes stored for prolonged periods at 28°C. Intra-thoracically inoculated mosquitoes with confirmed positivity were killed by quick freezing at -80°C, applied to sticky tape, and stored at 28°C with 80 ± 5% relative humidity (RH). At weekly intervals, five mosquitoes were removed from the tape randomly and assayed individually for detection of viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). CHIKV RNA was detected up to 12 weeks in dry mosquitoes by RT-PCR. Virus could not be isolated either in cell culture or in the suckling Swiss-albino mouse system at any stage. This study demonstrated the persistence of CHIKV viral RNA up to 12 weeks when stored at 28°C with RH 80 ± 5%. This finding will have significance in CHIKV surveillance programs in mosquito populations or field-based studies in countries where maintenance of a cold chain is a concern.

Venereal transmission of chikungunya virus by Aedes aegypti mosquitoes (Diptera: Culicidae).

Experiments were conducted to demonstrate the role of male Aedes aegypti mosquitoes in the maintenance and transmission of chikungunya virus (CHIKV) to female mosquitoes. We demonstrated that infected male mosquitoes are capable of infecting females during mating. The infection rate in female mosquitoes was 11% when virgin female mosquitoes were allowed to coinhabit with infected males. The body suspension of venereally infected female mosquitoes induced illness in infant Swiss albino mice, which demonstrated the infectivity of the venereally transmitted virus. The presence of CHIKV in the brains of the ill mice was confirmed by a reverse transcription-polymerase chain reaction specific for partial sequences of nonstructural protein 4 and envelope 1 genes. In the light of the recent report of transovarial transmission of CHIKV in mosquitoes, although at a lower level, this finding has significance because it may help in transmission of the virus to females venereally to start a new infection cycle.

Experimental transmission of Chandipura virus by Phlebotomus argentipes (diptera: psychodidae).

Experiments were carried out to demonstrate the susceptibity and transmission potential of Phlebotomus argentipes (Annandale & Brunetti) for Chandipura virus (CHPV). In India, P. argentipes is one of the predominant species found in many areas endemic for CHPV. Although its laboratory colonization is difficult, we have demonstrated that 65% of P. argentipes were susceptible to CHPV infection by the oral route. Transmission experiments were carried out by intrathoracic inoculation because of re-feeding problems with this species. After incubation for 24 hours, efficient transmission of CHPV to mice was observed. The estimated minimum transmission rate among the inoculated flies was 32%. CHPV in sand flies as well as in mice was detected and confirmed by immunofluorescent antibody assay and reverse transcription-polymerase chain reaction, respectively. The susceptibility of P. argentipes to CHPV and its potential to transmit the virus by bite has importance in epidemiology of CHPV.