Experiments were carried out to demonstrate the susceptibity and transmission potential of Phlebotomus argentipes (Annandale &amp; 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

Fulmali, Pradip V.

Geevarghese, G.

Ghodke, Yuvraj S.

Kanojia, Phoolchand

Mavale, Mangala S.

Mishra, Akhilesh C.

English

Publications of the IAS Fellows

EXPERIMENTAL TRANSMISSION OF CHANDIPURA VIRUS BY PHLEBOTOMUS
ARGENTIPES (DIPTERA: PSYCHODIDAE)
MANGALA S. MAVALE, PRADIP V. FULMALI, YUVRAJ S. GHODKE, AKHILESH C. MISHRA,
PHOOLCHAND KANOJIA, AND G. GEEVARGHESE*
National Institute of Virology, Pune, India
Abstract. 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 intra-thoracic 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 Ve-
siculovirus) 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 enceph-
alopathy occurred in Andhra Paradesh, India, and CHPV iso-
lated from a human cerebrospinal fluid sample.3 In India,
CHPV has been isolated from phlebotomine sand flies col-
lected at Aurangabad, Maharashtra.4 Transovarial transmis-
sion of CHPV in the laboratory has been reported for Phle-
botomus papatasi (Scopoli).5 Recently, CHPV has been de-
tected 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 transmis-
sion 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 aspi-
rator. 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 main-
tained 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 ap-
proximately 102 plaque-forming units (PFU) of CHPV (Na-
tional Institute of Virology strain no. 653514.). After expo-
sure 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 main-
tained 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, anes-
thetized 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. De-
spite repeated efforts, no re-feeding on mice was observed.
Thus, for further experimentation, we inoculated P. argenti-
pes 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 inocu-
lated 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
* Address correspondence to Gevaeghese Geevarghese, National
Institute of Virology, 20/A, Ambedkar Road, Pune-411001, India.
E-mail: geevarghese_niv@yahoo.co.in
Am. J. Trop. Med. Hyg., 76(2), 2007, pp. 307–309
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
307
feeding and extrinsic incubation by using an immunofluores-
cent 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 se-
rum 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 fe-
males 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 differ-
ent 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 demon-
strate transmission caused by individual flies. Instead, mini-
mal transmission rates were calculated by dividing the num-
ber of transmission events (i.e., number of mice becoming ill
after infection with CHPV) by the number of potential trans-
mitters (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 spe-
cies 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 condi-
tions.11,12
We showed that CHPV can multiply in P. argentipes and
can be transmitted by bite. This observation is significant be-
cause Phlebotomine sand flies have a broad host range that
includes humans and a wide range of mammals.13 An inter-
esting 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 ex-
tremely 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 trans-
mission 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 ex-
periments, 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 spe-
cies rests in cattle sheds found near areas of human habitation
and fed mostly on bovines.8 There were also reports of Phle-
botomine 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 in-
ability 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.
Received February 20, 2006. Accepted for publication September 13,
2006.
Acknowledgment: We are grateful to the entomology staff, especially
P. R. Salunke, for collection of sand flies and rearing them in the
laboratory.
Authors’ address: Mangala S. Mavale, Pradip V. Fulmali, Yuvraj S.
Ghodke, Akhilesh C. Mishra, Phoolchand Kanojia, and G. Geevargh-
ese, National Institute of Virology, 20/A, Ambedkar Road, Pune-
411001, India.
REFERENCES
1. Bhatt PN, Rodrigues FM, 1967. Chandipura virus: a new arbovi-
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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.
TABLE 1
Experimental infection rate and transmission of Chandipura virus
(CHPV) by Phlebotomus argentipes sand flies*
Method of infection
Total no. of flies
feeding/total no.
of flies tested
Viral
dissemination
rate
Minimum
transmission rate†
Orally exposed
sand flies 17/30 65% Not determined
Inoculated sand flies 19/30 93% 32%
† Total number of CHPV-positive mice/total number of inoculated flies that engorged.
MAVALE AND OTHERS308
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
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TRANSMISSION OF CHANDIPURA VIRUS BY P. ARGENTIPES 309


Experimental transmission of Chandipura virus by Phlebotomus argentipes (Diptera: Psychodidae)

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Experimental transmission of Chandipura virus by Phlebotomus argentipes (Diptera: Psychodidae)

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