• 1

    Bhatt PN, Rodrigues FM, 1967. Chandipura virus: a new arbovirus isolated in India from patient with febrile illness. Indian J Med Res 55 :1295–1305.

    • Search Google Scholar
    • Export Citation
  • 2

    Rodrigues FM, Patankar MR, Banerjee K, Bhatt PN, Goverdhan MK, Pavri KM, Vittal M, 1972. Etiology of the 1965 epidemic of febrile illness in Nagpur city, Maharashtra state, India. Bull World Health Organ 46 :173–179.

    • Search Google Scholar
    • Export Citation
  • 3

    Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA, Thakare JP, Ramesh JD, Rao KA, Mishra AC, 2004. A large outbreak of acute encephalitis with high case fatality rate in children in Andhra Pradesh, India in 2003 associated with Chandipura virus. Lancet 4–10 :869–874.

    • Search Google Scholar
    • Export Citation
  • 4

    Dhanda V, Rodrigues FM, Ghosh SN, 1972. Isolation of Chandipura virus from sand flies in Aurangabad. Indian J Med Res 58 :179–180.

  • 5

    Tesh RB, Modi GB, 1983. Growth and transovarial transmission of Chandipura virus (Rhabdoviridae: Vesiculovirus) in Phlebotomus papatasi. Am J Trop Med Hyg 32 :621–623.

    • Search Google Scholar
    • Export Citation
  • 6

    Geevarghese G, Arankalle VA, Jadi R, Kanojia PC, Joshi MV, Mishra AC, 2005. Detection of Chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India. J Med Entomol 42 :495–496.

    • Search Google Scholar
    • Export Citation
  • 7

    Hardy JL, 1988. Susceptibility and resistance of vector mosquitoes, Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Volume II. Boca Ration, FL: CRC Press, 87–126.

  • 8

    NIV Annual Reports, 2004. Pune, India: National Institute of Virology.

  • 9

    Lewis DJ, 1967. The phlebotomine sand-flies of west Pakistan (Diptera-Psychodidae. Bull Br Mus 19 :1–57.

  • 10

    Kuberski TT, Rosen L, 1977. A simple technique for the detection of dengue antigen in mosquitoes by immunofluorescence. Am J Trop Med Hyg 26 :533–537.

    • Search Google Scholar
    • Export Citation
  • 11

    Hertig M, Jahnson PT, 1961. The rearing of Phlebotomus sand flies. I. Technique. Ann Entomol Soc Am 54 :753–764.

  • 12

    Jahnson PT, Hertig M, 1961. The rearing of Phlebotomus sand flies. II. Development and behavior of Panamanian sand flies in laboratory culture. Ann Entomol Soc Am 54 :764–776.

    • Search Google Scholar
    • Export Citation
  • 13

    Tesh RB, Chaniotis BN, Aronson M, Johnson KM, 1971. Natural host preferences of Panamanian Phlebotomine sand flies as determined by precipitation test. Am J Trop Med Hyg 20 :150–156.

    • Search Google Scholar
    • Export Citation
  • 14

    Ilkal MA, Goverdhan MK, Shetty PS, Tupe CD, Mavale MS, Dhanda V, 1991. Susceptibility of four species of mosquitoes to Chandipura virus and its detection by immunofluorescence. Acta Virol 35 :27–32.

    • Search Google Scholar
    • Export Citation
  • 15

    Schmidt JR, Schmidt ML, 1965. Observation on feeding habits of Phlebotomus papatasi under stimulated natural condition. J Med Entomol 2 :225–230.

    • Search Google Scholar
    • Export Citation

 

 

 

 

EXPERIMENTAL TRANSMISSION OF CHANDIPURA VIRUS BY PHLEBOTOMUS ARGENTIPES (DIPTERA: PSYCHODIDAE)

View More View Less
  • 1 National Institute of Virology, Pune, India

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.

INTRODUCTION

Chandipura virus (CHPV, family Rhabdoviridae, genus Vesiculovirus) was isolated originally from human serum during an outbreak of febrile illness in Nagpur, Maharashtra, India in 1965.1,2 Recently, an outbreak of febrile illness with encephalopathy occurred in Andhra Paradesh, India, and CHPV isolated from a human cerebrospinal fluid sample.3 In India, CHPV has been isolated from phlebotomine sand flies collected at Aurangabad, Maharashtra.4 Transovarial transmission of CHPV in the laboratory has been reported for Phlebotomus papatasi (Scopoli).5 Recently, CHPV has been detected in sergentomine sand flies.6 Therefore, sand flies are considered to be main vector of CHPV.

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication, and transmission of a virus.7 When an arthropod vector takes a viremic blood meal, the virus has to cross two physiologic barriers: 1) midgut barriers for viral infection and dissemination into the hemocoel, and 2) salivary gland barriers for viral secretion and transmission by bite.

Among Phebotomine sand flies, P. argentipes is one of the predominant species in several regions of India, especially in the CHPV-epidemic areas of Warangal and Chotta Udaypur, Gujarat, India.8 The purpose of our study was to demonstrate the susceptibility and transmission potential of P. argentipes for CHPV.

MATERIALS AND METHODS

Collection and rearing of sand flies.

Wild sand flies were collected in cattle sheds and human dwellings by hand aspirator. The next morning, flies were placed in a cloth feeding cage. Two mice (5–6 days old) were then introduced in the cage. After feeding, blood-engorged flies were removed from the cage and were placed individually in specially designed plastic jars with plaster of paris at the bottom and were maintained at 28–30°C. After oviposition, females were removed from the jar and were used for subsequent identification up to the species level by using the key of Lewis.9 Emerging larvae from the deposited eggs were fed with larval food comprising of sand, cow dung, and goat liver powder in the ratio of 55: 40:5. The emerging adult females of P. argentipes (F1) were used for susceptibility and transmission experiments.

Oral infection and viral dissemination.

The F1 females were removed from the jars and placed in a cloth feeding cage and starved for 24 hours. In three separate trials, two viremic mice (2–3 days old) were introduced into the cage. These mice were inoculated intraperitoneally 24 hours earlier with approximately 102 plaque-forming units (PFU) of CHPV (National Institute of Virology strain no. 653514.). After exposure to sand flies for three hours, mice were removed from the feeding cage and bled. Blood was diluted 1:10 in phosphate-buffered saline solution and stored in freezer at −70°C for subsequent virus titration. All visibly blood-engorged flies were removed from the cages and put in small plastic jars, which were then covered with muslin cloth and set in a pan. These sand flies were provided daily with glucose as the sources of carbohydrate and housed in an insectary maintained between 28°C and 30°C at a relative humidity of 95% and with 12 hours of light per day for 4–5 days. After extrinsic incubation, these sand flies were collected in test tubes, anesthetized at −70°C, and head squashes were prepared. The bodies were placed in sterile screw-capped vials and stored at −70°C for further virus detection.

Viral transmission by bite.

The re-feeding of the flies was the major problem faced in transmission experiments. Despite repeated efforts, no re-feeding on mice was observed. Thus, for further experimentation, we inoculated P. argentipes females intrathoracically with CHPV. Transmission of CHPV was demonstrated in three separate trials by exposing two newborn Swiss-Albino mice (2–3 days of age) to inoculated sand flies (n = 10) 24 hours after inoculation. Mice were exposed in a feeding cage overnight. At the end of this period, these sand flies were killed and stored at −70°C for virus detection. The exposed mice were then returned to their mother and examined daily for infection. The sick and dying mice were killed and brains were harvested and triturated. The resultant suspension was used for viral RNA detection by reverse transcription–polymerase chain reaction (RT-PCR).

Virus detection and confirmation.

The disseminated CHPV was detected in brain cells of the fed sand flies after oral feeding and extrinsic incubation by using an immunofluorescent antibody assay following the procedure of Kuberski and Rosen10 and further confirmed by RT-PCR following the methods of Geevarghese and others3 and Rao and others.6 Head squashes of the P. argentipes were prepared on slides and processed to detect viral antigen using hyperimmune serum raised against the CHPV virus in mice. The bodies of these flies were triturated in 1 mL of sterile 0.75% bovine albumin phosphate saline. The resulting suspension was then inoculated intraperitoneally into mice. The harvested brains of the sick and dying mice were processed by RT-PCR to detect the viral RNA by using CHPV G protein–specific primers. The CHPV transmitted by bite was detected in the brains of the sick mice by using RT-PCR.

RESULTS

Sand flies collection and rearing.

A total of 178 (97 female and 81 male) sand flies were collected, of which 74 (48 females and 26 males) were identified as P. argentipes. Feeding and egg laying were recorded in 20–25% of the flies. A total of 390 eggs were collected and kept for hatching in six different plastic jars. A total of 148 (68 females and 80 males) adults emerged with female-to-male ratio of 1:1.17.

Oral infection and viral dissemination.

Three groups of 10 females were fed on viremic mice (titer = approximately 106 PFU). Feeding was observed in 40–70% of the females. After feeding, these females were examined after 4–5 days. Among the 17 fed females, 11 (65%) had disseminated virus in brain cells (Table 1).

Viral transmission by bite.

Three groups of 10 female flies each were inoculated intrathoracically and kept overnight in three different cages, with each cage containing a pair of baby mice. Fly feeding rates in the three groups were 60%, 60%, and 70% (mean = 63%, Table 1). Viral dissemination rates in the three groups of flies were 100%, 100%, and 80% (mean = 93%, Table 1). Because pairs of mice were each exposed to 10 inoculated flies, it was not possible to demonstrate transmission caused by individual flies. Instead, minimal transmission rates were calculated by dividing the number of transmission events (i.e., number of mice becoming ill after infection with CHPV) by the number of potential transmitters (i.e., number of blood-fed sand flies in the cages). Minimum transmission rates in the three groups were 33%, 33%, and 29% (mean = 32%, Table 1).

DISCUSSION

The major constraints faced during colonization of this species were high mortality after oviposition and no females could survive longer than 8–9 days. Approximately 25% of the wild-caught females and 60–70% of the F1 Ph. argentipes females could feed during the initial exposure to mice but not on hamsters. No wild-caught or F1 generation females of this species would re-feed on these host on re-exposure. Our present studies confirm earlier reports that the Phlebotomine sand flies are difficult to colonize in laboratory conditions.11,12

We showed that CHPV can multiply in P. argentipes and can be transmitted by bite. This observation is significant because Phlebotomine sand flies have a broad host range that includes humans and a wide range of mammals.13 An interesting feature of this study was that P. argentipes was able to transmit CHPV to newborn mice by bite.

Our study showed that P. argentipes is susceptible to CHPV infection and the virus is disseminated to salivary gland by overcoming the midgut barrier and midgut escape barrier within 4–5 days and is then transmitted to other vertebrate hosts by crossing salivary gland barrier in the next 24 hours. This indicates that the extrinsic incubation period of CHPV in P. argentipes is approximately 5–6 days. In P. papatasi and some mosquito species, it was shown that CHPV has an extremely short extrinsic incubation period.5,14 Although we were are not able to identify the precise incubation period because of the re-feeding problem, it appears that CHPV has short extrinsic incubation period that may favor rapid transmission of CHPV.

To ensure efficient transmission of the virus by the vector species, rapid virus multiplication in vectors and its proximity to human habitation would seem to be essential. In our experiments, we have shown that the CHPV can multiply in P. argentipes and that this species can transmit CHPV by bite. Distribution data for sand flies showed that P. argentipes is one of the predominant species found in several parts of India endemic for CHPV.8 It has also been reported that this species rests in cattle sheds found near areas of human habitation and fed mostly on bovines.8 There were also reports of Phlebotomine sand flies re-feeding and some can re-feed as often as every two days.15

Our inability to demonstrate CHPV transmission by the orally infected P. argentipes probably resulted from the inability of P. argentipes to re-feed. However, the ability of P. argentipes to acquire CHPV from a viremic host and transmit it has immense epidemiologic importance.

Table 1

Experimental infection rate and transmission of Chandipura virus (CHPV) by Phlebotomus argentipes sand flies*

Method of infectionTotal no. of flies feeding/total no. of flies testedViral dissemination rateMinimum transmission rate†
† Total number of CHPV-positive mice/total number of inoculated flies that engorged.
Orally exposed sand flies17/3065%Not determined
Inoculated sand flies19/3093%32%

*

Address correspondence to Gevaeghese Geevarghese, National Institute of Virology, 20/A, Ambedkar Road, Pune-411001, India. E-mail: geevarghese_niv@yahoo.co.in

Authors’ address: Mangala S. Mavale, Pradip V. Fulmali, Yuvraj S. Ghodke, Akhilesh C. Mishra, Phoolchand Kanojia, and G. Geevarghese, National Institute of Virology, 20/A, Ambedkar Road, Pune-411001, India.

Acknowledgment: We are grateful to the entomology staff, especially P. R. Salunke, for collection of sand flies and rearing them in the laboratory.

REFERENCES

  • 1

    Bhatt PN, Rodrigues FM, 1967. Chandipura virus: a new arbovirus isolated in India from patient with febrile illness. Indian J Med Res 55 :1295–1305.

    • Search Google Scholar
    • Export Citation
  • 2

    Rodrigues FM, Patankar MR, Banerjee K, Bhatt PN, Goverdhan MK, Pavri KM, Vittal M, 1972. Etiology of the 1965 epidemic of febrile illness in Nagpur city, Maharashtra state, India. Bull World Health Organ 46 :173–179.

    • Search Google Scholar
    • Export Citation
  • 3

    Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA, Thakare JP, Ramesh JD, Rao KA, Mishra AC, 2004. A large outbreak of acute encephalitis with high case fatality rate in children in Andhra Pradesh, India in 2003 associated with Chandipura virus. Lancet 4–10 :869–874.

    • Search Google Scholar
    • Export Citation
  • 4

    Dhanda V, Rodrigues FM, Ghosh SN, 1972. Isolation of Chandipura virus from sand flies in Aurangabad. Indian J Med Res 58 :179–180.

  • 5

    Tesh RB, Modi GB, 1983. Growth and transovarial transmission of Chandipura virus (Rhabdoviridae: Vesiculovirus) in Phlebotomus papatasi. Am J Trop Med Hyg 32 :621–623.

    • Search Google Scholar
    • Export Citation
  • 6

    Geevarghese G, Arankalle VA, Jadi R, Kanojia PC, Joshi MV, Mishra AC, 2005. Detection of Chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India. J Med Entomol 42 :495–496.

    • Search Google Scholar
    • Export Citation
  • 7

    Hardy JL, 1988. Susceptibility and resistance of vector mosquitoes, Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Volume II. Boca Ration, FL: CRC Press, 87–126.

  • 8

    NIV Annual Reports, 2004. Pune, India: National Institute of Virology.

  • 9

    Lewis DJ, 1967. The phlebotomine sand-flies of west Pakistan (Diptera-Psychodidae. Bull Br Mus 19 :1–57.

  • 10

    Kuberski TT, Rosen L, 1977. A simple technique for the detection of dengue antigen in mosquitoes by immunofluorescence. Am J Trop Med Hyg 26 :533–537.

    • Search Google Scholar
    • Export Citation
  • 11

    Hertig M, Jahnson PT, 1961. The rearing of Phlebotomus sand flies. I. Technique. Ann Entomol Soc Am 54 :753–764.

  • 12

    Jahnson PT, Hertig M, 1961. The rearing of Phlebotomus sand flies. II. Development and behavior of Panamanian sand flies in laboratory culture. Ann Entomol Soc Am 54 :764–776.

    • Search Google Scholar
    • Export Citation
  • 13

    Tesh RB, Chaniotis BN, Aronson M, Johnson KM, 1971. Natural host preferences of Panamanian Phlebotomine sand flies as determined by precipitation test. Am J Trop Med Hyg 20 :150–156.

    • Search Google Scholar
    • Export Citation
  • 14

    Ilkal MA, Goverdhan MK, Shetty PS, Tupe CD, Mavale MS, Dhanda V, 1991. Susceptibility of four species of mosquitoes to Chandipura virus and its detection by immunofluorescence. Acta Virol 35 :27–32.

    • Search Google Scholar
    • Export Citation
  • 15

    Schmidt JR, Schmidt ML, 1965. Observation on feeding habits of Phlebotomus papatasi under stimulated natural condition. J Med Entomol 2 :225–230.

    • Search Google Scholar
    • Export Citation
Save