Vector competence of Aedes vittatus (Bigot) mosquitoes from India for Japanese encephalitis, West Nile, Chandipura and Chittoor viruses.

BACKGROUND & OBJECTIVES: Aedes vittatus (Bigot), an anthropophilic mosquito, plays an important role in the maintenance and transmission of yellow fever (YF), dengue (DEN), chikungunya (CHIKV) and Zika (ZIK) viruses in Africa. In India, though natural isolation of none of these viruses was reported from the mosquito, experimental studies have shown vector competence to DEN and CHIK viruses. Despite wide prevalence in India, their potential in transmitting viruses of public health importance viz., Japanese encephalitis (JEV), West Nile (WNV), Chandipura (CHPV), Chittoor (CHITV) etc., has never been investigated. The objective of the present study is to determine the vector potential of the mosquito to these viruses. METHODS: Mosquitoes were infected by intra-thoracic inoculation as well as by oral feeding, and growth kinetics was determined. Virus dissemination to organs was investigated by determining virus in the harvested organs on specified days' post infection (PI). Vector competence was determined by detecting the virus in saliva. RESULTS: Intra thoracic inoculation has shown vector competence of the mosquito to JEV, WNV, CHIV and CHPV. However, using the oral route of infection, replication was observed with only WNV, JEV and CHITV. High degree of WNV replication (6.7log TCID50/ml) with rapid dissemination to wings, legs and salivary glands was seen from 5th day PI onwards. WNV was detected in saliva with a titer of 0.7log10 TCID50/ml on 5th day PI. JEV and CHITV replicated in the mosquito yielding 3log and 4log10 TCID50/ml on 5th and 10th day PI respectively, but virus was not detected in saliva till 15th day PI. INTERPRETATION & CONCLUSION: From the results it is difficult to indict the mosquito as a vector of the viruses studied. However, presence of WNV in saliva of the mosquito shows its potential as a bridge vector and poses a concern especially when virulent WNV strains are circulating in the country.

Temperature sensitivity and environmental stability of Chandipura virus.

Chandipura virus (CHPV), a negative-stranded RNA virus of family Rhabdoviridae is endemic in Central India since 1965. The virus gained public health importance when it was held responsible for massive outbreak in 2003-2004 in Maharashtra, Telengana and Gujarat with case fatality rates ranging from 55 to 75% among children. We studied the stability of the virus as well as RNA persistence in samples stored at different temperatures for different periods. CHPV remained infective in sand flies and cell culture supernatants at 4 °C for 8 weeks. At 37 °C CHPV remained viable for 18 days when stored in infected cell supernatant (Minimum essential medium supplemented with 10% fetal bovine serum). However, in infected sand flies stored at 37 °C, the virus lost virulence within a week. CHPV RNA, though lost virulence, could be detected in virus exposed sand flies stored at 37 °C for 13 weeks by real time RT-PCR. Retaining virulence at 37 °C for 18 days in serum containing medium is a matter of concern for laboratories and hospital settings where clinical samples are handled. RNA persistence for prolonged periods in dead sand flies might help in surveillance studies of CHPV in sand flies and will help in resource constraint nations where cold chain management is a concern.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Vectorial capacity of Culex gelidus (Theobald) mosquitoes to certain viruses of public health importance in India.

BACKGROUND & OBJECTIVES: Culex gelidus, a widely prevalent mosquito in India and Southeast Asia region, is an important vector of Japanese encephalitis virus (JEV). Experimental studies have shown its potential to transmit West Nile, Kunjin, Murray Valley encephalitis and Ross River viruses. An attempt was therefore made to study its susceptibility and vector competence to some of the arboviruses of public health importance in India. METHODS: Mosquitoes were infected with six viruses, viz. JEV, chikungunya (CHIKV), Chandipura (CHPV), Chittoor (CHITV), Ingwavuma (INGV) and Umbre (UMBV) by intra thoracic inoculation to determine virus susceptibility and vector competence. Growth kinetics of the viruses were studied by determining the titres of inoculated mosquitoes on different days post-infection by titration in Vero E6 cells. Vector competence was studied by detecting the presence of the viruses in saliva of infected mosquitoes. RESULTS: All the six viruses were replicated in Cx. gelidus. JEV, CHPV, CHIKV and CHITV yielded > 5 log10TCID50/ml virus while UMBV and INGV yielded approx 4log10TCID50/ml virus. JEV, CHIKV and CHITV could be detected in the saliva of the infected mosquitoes, while CHPV, INGV and UMBV could not be detected in the saliva of the infected mosquitoes. INTERPRETATION & CONCLUSION: Replication potential and vector competence of Cx. gelidus to some of the viruses of public health importance in India, viz. JEV, CHIKV, CHITV etc, pose a serious threat to general population, especially in the wake of spurt in its population in certain parts of India.

Bagaza virus inhibits Japanese encephalitis & West Nile virus replication in Culex tritaeniorhynchus & Cx. quinquefasciatus mosquitoes.

BACKGROUND & OBJECTIVES: Studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting/enhancing replication of important flaviviruses. Hence, a study was designed to determine whether Bagaza virus (BAGV), a flavivirus isolated from Culex tritaeniorhynchus mosquitoes in India, alters susceptibility of Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes to Japanese encephalitis (JEV) and West Nile viruses (WNV). METHODS: JEV and WNV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes in the presence of BAGV was carried out by intrathoracic (IT) inoculation and oral feeding methods. Mosquitoes were infected with BAGV and WNV/JEV either simultaneously or in a phased manner, in which mosquitoes were infected with BAGV by IT inoculation followed by super-infection with JEV/WNV after eight days post-infection (PI). JEV and WNV yield on 7 [th] and 14 [th] day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID 50 ) method. RESULTS: In Cx. tritaeniorhynchus mosquitoes, prior infection with BAGV significantly reduced JEV and WNV replication while in Cx. quinquefasciatus, BAGV influence was only seen with WNV. Reduction in virus titre was observed in IT inoculated and oral fed mosquitoes irrespective of the infection mode. JEV replication was also found reduced in Cx. tritaeniorhynchus mosquitoes persistently infected with BAGV at passage four. INTERPRETATION & CONCLUSIONS: BAGV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes altered their susceptibility to JEV and WNV producing low virus yield. However, the role of BAGV in inhibiting JEV/WNV replication in field mosquitoes needs further investigations.

Replication potential and different modes of transmission of West Nile virus in an Indian strain of Culex gelidus Theobald (Diptera: Culicidae) mosquitoes.

BACKGROUND & OBJECTIVES: Culex gelidus mosquito, an important vector of Japanese encephalitis virus, has shown to transmit West Nile virus (WNV), Kunjin and Murray Valley encephalitis viruses experimentally. An attempt was, therefore, made to study the replication kinetics and vector competence of an Indian strain of Cx. gelidus to WNV. METHODS: Mosquitoes were infected by both intrathoracic inoculation and oral feeding and studied the growth kinetics by determining the virus titre on different days post-infection (PI). Vector competence was studied by determining the presence of WNV in saliva on subsequent days PI. Horizontal transmission was determined by demonstrating infection in infant mice by bite of mosquitoes that were fed on viraemic mice previously. Vertical transmission was studied by screening progeny derived from infected mosquitoes. Trans-stadial transmission was determined by screening adult mosquitoes emerged from parenterally inoculated IV instar larvae. RESULTS: The mosquito replicated WNV to 7log10 TCID50/ml on Day 8 PI and maintained the titre for 14 days. Virus dissemination to legs and salivary glands could be detected, but not to ovaries up to Day 10 PI. The mosquitoes picked up infection from viraemic blood and transmitted successfully to infant mice on subsequent feeding. Trans-stadial transmission also could be demonstrated. However, vertical transmission could not be demonstrated. INTERPRETATION & CONCLUSION: The replication potential, maintenance of WNV for prolonged periods and ability to transmit WNV experimentally makes the mosquito a serious threat to public health especially in the wake of active WNV activity in certain parts of India.

Preliminary findings on Bagaza virus (Flavivirus: Flaviviridae) growth kinetics, transmission potential & transovarial transmission in three species of mosquitoes.

BACKGROUND & OBJECTIVES: Bagaza virus (BAGV), a flavivirus synonymous with Israel turkey meningoencephalitis virus, has been found to circulate in India. BAGV has recently been held responsible for inducing febrile illness in humans and causing unusually high mortality to wild birds in Spain. A study was therefore, undertaken to determine its replication kinetics in certain mosquitoes and to determine vector competence and potential of the mosquitoes to transmit BAGV experimentally. METHODS: Aedes aegypti, Culex tritaeniorhynchus and Cx quinquefasciatus mosquitoes were inoculated with BAGV; samples were harvested every day and titrated in BHK-21 cell line. Vector competence and experimental transmission were determined by examining the saliva of infected mosquitoes for virus and induction of sickness in suckling mice, respectively. RESULTS: Cx. tritaeniorhynchus and Ae. aegypti mosquitoes yielded 5 log10 and 4.67 log10 TCID50/ml of virus on day 3 post-infection (PI), respectively while Cx. quinquefasciatus yielded a titre of 4 log10 TCID50/ml on day 4 PI. BAGV was detected in saliva of all the infected mosquitoes demonstrating their vector competence. Experimental transmission of BAGV to infant mice as well as transovarial transmission was demonstrated by Cx. tritaeniorhynchus but not by Ae. aegypti and Cx. quinquefasciatus mosquitoes. INTERPRETATION & CONCLUSIONS: Replication of BAGV to high titres and dissemination to saliva in three most prevalent mosquitoes in India is of immense public health importance. Though no major outbreak involving man has been reported yet, BAGV has a potential to cause outbreaks in future.

Venereal transmission of Chandipura virus by Phlebotomus papatasi (Scopoli).

Experiments were conducted in the laboratory on Phlebotomus papatasi to determine the possible role of males in maintaining or sustaining the Chandipura virus (CHPV) activity in nature. This study indicated that infected males are capable of passing on the virus to female sand flies while mating. The infection rate was found to be 12.5% in uninfected females when mated with infected males. The occurrence of venereal transmission of this virus may have epidemiologic importance in the natural cycle of CHPV.

Vertical and venereal transmission of Chandipura virus (Rhabdoviridae) by Aedes aegypti (Diptera: Culicidae).

Experiments in the laboratory documented vertical and venereal transmission of Chandipura virus (CHPV) in Aedes aegypti (L.). The minimum filial infection rate among the progeny of infected females was 1.2%; the rate among male and female progeny was 0.9 and 1.4%, respectively. The venereal infection rate of CHPV among inseminated females was 32.7%. Our study indicates the possible occurrence of vertical and venereal transmission of CHPV in insect vectors.

Isolation of Ganjam virus from ticks collected off domestic animals around Pune, Maharashtra, India.

Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.