Canadian Journal of Microbiology
Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of Batai virus
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2017-0514.R2
Manuscript Type: Article
Date Submitted by the Author: 02-Apr-2018
Complete List of Authors: Sudeep, Anakkathil; National Institute of Virology, Medical Entomology & Zoology Shaikh, Neda; National Institute of Virology Ghodke, Youwaraj;Draft National Ingale, Viswas; National Institute of Virology Gokhale, Mangesh; National Institute of Virology
Is the invited manuscript for consideration in a Special N/A Issue? :
Batai virus, Chittoor virus, Culex quinquefasciatus, Culex tritaeniorhynchus, Keyword: vector competence
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Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of Batai virus
Sudeep AB, Neda Shaikh, Ghodke YS, Ingale VS, Gokhale MD ICMR National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan PUNE –411021, India.
Draft
Running head: Chittoor virus
Address for correspondence:
Dr. AB Sudeep, ICMR National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, PUNE 411021. Tel.91 20 26006390, Fax.91 20 25871895. E-mail: [email protected].
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Abstract
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 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 log10TCID50/ml, respectively, and maintained the virus for substantial periods. Both the Culex species demonstrated vectorDraft competence, while Ae aegypti did not. Horizontal transmission to infant mice was also demonstrated by both the 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.
Key words: Batai virus, Chittoor virus, Culex quinquefasciatus, Culex tritaeniorhynchus, vector competence.
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Introduction
Chittoor virus (CHITV) is a negative sense, single stranded RNA virus belonging to
genus Orthobunyavirus of the family Bunyaviridae, which comprises highly pathogenic
viruses, i.e., Crimean Congo hemorrhagic fever virus, Hantan virus, Rift Valley fever virus,
and Nairobi sheep disease virus. CHITV was first isolated from Anopheles barbirostris
mosquitoes collected from the Chittoor district of Andhra Pradesh, India in 1957 during an
investigation of arthropod borne viruses in mosquitoes. Subsequent studies yielded several
isolations of CHITV from different species of mosquitoes and once from a piglet (Table 1).
Seroprevalence studies have shown the presence of neutralizing antibodies in human and
vertebrate sera collected from different parts of the country, demonstrating the circulation of
CHITV or a closely related virus in IndiaDraft since 1957 or earlier (Table 2).
Serological studies have revealed the antigenic closeness of CHITV to the Batai virus
(BATV) and the Colovo virus isolated from Malaysia and Czechoslovakia, respectively
(Singh and Pavri 1966; Pavri and Sheikh 1966). BATV (genus Orthobunyavirus) has a wide
distribution in Asia, Africa and Europe and causes influenza like illness in humans and mild
illness in sheep and goats (Hoffman et al. 2015).
Bunyaviruses are known for genetic reassortment in nature, wherein they reshuffle
genomic segments between members of the same serogroup, thereby altering their
pathogenicity (Yadav et al. 2012; Brown et al. 2001). Co infection of a host cell with
multiple viruses may result in the shuffling of gene segments to generate progeny viruses
with novel genome combinations. The Ngari virus, a reassortant virus formed with the S and
L segments of the Bunyamwera virus and the M segment of BATV caused hemorrhagic fever
in humans with devastating effects in East Africa (Briese et al. 2006). Further, a subtype of
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BATV, the Ilesha virus, has also been associated with hemorrhagic fever in Africa (Briese et al. 2006; Gerrard et al. 2004).
Although no outbreak of CHITV involving humans or other vertebrates has been reported in India yet, the presence of antibodies in humans is indicative of subclinical infection. The virus has been isolated repeatedly from different species of mosquitoes, demonstrating its maintenance principally in mosquitoes. However, no studies have been carried out either to determine the vector competence of these mosquitoes or to understand the maintenance of the virus in nature, which may be due to its low pathogenicity. In the present communication, we report on the competence of Culex quinquefasciatus, Cx tritaeniorhynchus and Aedes aegypti mosquitoes to maintain and horizontally transmit CHITV under experimental conditionsDraft in the laboratory. This study is relevant because these mosquitoes are widely distributed and common human biting species in India where this virus and other bunyaviruses circulate.
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Materials and Methods
Place of study: The study was conducted in the Department of Medical Entomology and
Zoology, Microbial Containment Complex, National Institute of Virology (NIV), Pashan,
Pune, India.
Virus strain:
Strain number 804992 (Acc. Nos. FJ436796 L FJ436801 M FJ436805 N) isolated from
Culex pseudovishnui mosquitoes from Karnataka in 1980 (NIV unpublished data) was used in
the study. The strain has been passaged 4 times through the Vero cell line.
Mosquitoes:
Mosquitoes were procured from the insectary maintained by the Entomology division of NIV, Pashan. Mosquito larvae were Draftfed on commercially available fish food, while adults were maintained on a diet of 10% glucose. Female mosquitoes were provided with 2 to 3
month old fowl for blood meals on alternate days. Virus exposed mosquitoes were kept in
plastic jars inside mosquito cages, and all the experiments were carried out inside a bio safety
level 2 laboratory, which has containment facilities to prevent the escape/entry of mosquitoes.
Virus exposed and unexposed mosquitoes were maintained at 28±2°C with 80±5% relative
humidity and a 12:12 hr light: dark regime.
Ethical clearance to conduct animal experiments: The work was approved by the
institutional animal ethical committee, and all the experiments were carried out in accordance
with their recommendations.
Growth kinetics of CHITV in mosquitoes:
Three to four day old female mosquitoes of each species that were maintained on a
10% glucose diet (n=80) were fed orally for each experiment as described by Sudeep et al.
(2014). In brief, mosquitoes were starved for 4 6 hr and then allowed to feed on CHITV
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infected mice showing acute sickness (mice were inoculated intra cerebrally with CHITV three days prior to mosquito feeding). Fully engorged mosquitoes were separated, held in mosquito holding jars and secured in a double walled mosquito cage. The virus exposed mosquitoes were incubated at 28±2°C with 70 80% humidity and maintained on a 10% glucose diet. Ten mosquitoes of each species were harvested on alternate days from the ‘0’ day to the 12th day post infection (PI); 5 mosquitoes were stored at 80°C for virus titer determination and five mosquitoes for virus dissemination studies.
After completion of the experiment, the stored batches of mosquitoes of each species of each harvesting day were processed for determination of virus titer. Individual pools (n=5) were triturated in 1 ml of minimum essential medium (MEM, Invitrogen, USA) containing 2%
FBS using a hand held battery operated homogenizer (Sigma, USA). The suspension was clarified by centrifugation at 4°C forDraft 30 min at 5000 rpm, filtered (pore size=0.22 µm), diluted serially (10 fold) and titrated in Vero E6 cells in quadruplicate, as described earlier
(Sudeep et al. 2014). Virus titers were determined as described by Reed and Muench (1938).
The experiment was carried out in triplicate and analyzed to determine the viral growth kinetics.
Harvesting of wings, legs and saliva and determination of virus titer:
The extraction of saliva was carried out as described by van den Hurk et al. (2011). In brief, the wings of the mosquito were clipped (pooled wings of 5 mosquitoes and stored for virus titration), its proboscis was inserted into a capillary containing FBS and allowed to excite for
30 min. Contents of the capillary were transferred to 0.5 ml of chilled MEM (saliva from 5 mosquitoes) before being filtered (0.22 µm, Millipore) and titrated in Vero E6 cell line, as described above. After saliva extraction, legs of each mosquito were clipped gently, pooled
(legs of 5 mosquitoes), and stored for virus titer determination. Virus titer of pooled wings and legs of alternate day samples were determined as described above for whole body.
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Determination of horizontal and vertical transmission of CHITV:
Horizontal and vertical transmission of CHITV in mosquitoes was determined as described
by Sudeep et al. (2014). For horizontal transmission, a fresh batch of 3 4 day old mosquitoes
(n=50) were fed on viraemic mice and then the fully engorged mosquitoes were separated,
incubated for 11 days at 28°C and allowed to feed on 2 day old infant mice (n=8). Mice were
observed for sickness, the brains were harvested from the sick mice, triturated in MEM, and
inoculated into C6/36 cells after filtration (0.22 µm), and the presence of CHITV was
confirmed by the immunofluorescent antibody technique (IFA) on the 2nd day PI. The virus
titers of the brain suspensions were also determined by titration in Vero E6 cells.
For determination of vertical transmission, the virus exposed mosquitoes of each
species used for horizontal transmission study after infecting the mice were allowed to
oviposit and the F1 generation adults Draftwere screened for the presence of CHITV by IFA.
Results
All three species of mosquitoes were found to be susceptible for CHITV. Rapid
replication of the virus was observed from the initial day to the 4th day PI and the titer was
maintained throughout the study period. The maximum virus yield was observed in the Cx
quinquefasciatus species. Cx tritaeniorhynchus and Ae aegypti mosquitoes yielded lower
titers in comparison. The growth kinetics of CHITV in the respective mosquito species are
given below.
Cx quinquefasciatus mosquitoes:
CHITV growth kinetics in Cx quinquefasciatus mosquitoes showed rapid increase in virus
replication, with an approximately three fold increase in the virus titer on the 4th day PI (6.3
log10 TCID50/ml). Though a decrease in the titer was observed on subsequent days PI, the
mosquito maintained the virus for 12 days with a titer of >5 log TCID50/ml (Fig. 1).
Cx tritaeniorhynchus mosquitoes:
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The growth kinetics of CHITV in Cx tritaeniorhynchus mosquitoes was similar to those in Cx quinquefasciatus. The mosquitoes also showed rapid replication until the 4th day PI, yielding
5 log10TCID50/ml and maintained the titer throughout the study period (Fig. 1).
Aedes aegypti mosquitoes:
CHITV replication in Aedes aegypti was less rapid in comparison with the two Culex species (Fig. 1). The maximum virus yield was observed on the 4th day PI with a titer of 5.2 log10TCID50/ml, and the titer was maintained throughout the study period.
Virus dissemination to legs:
CHITV dissemination to legs was observed in the two Culex species from the 2nd day PI. A progressive increase in virus titer was observed on subsequent days PI (Fig. 2). Cx quinquefasciatus mosquitoes showed rapid dissemination and maximum virus yield in comparison to Cx tritaeniorhynchus Draft mosquitoes. After the 10th day PI, the virus yield was found identical in both mosquitoes. Virus dissemination to legs of virus exposed Aedes aegypti mosquitoes was not determined in the present study.
Virus dissemination to wings: None of the mosquitoes showed the presence of CHITV in the wings until the 12th day PI.
Vector competence of the mosquitoes:
Both the Culex mosquito species were found competent to transmit the virus, as CHITV was detected in their saliva from the 6th day PI. The virus yields on the 6th day PI in the saliva of
Cx quinquefasciatus and Cx tritaeniorhynchus were approximately 2 and 1.2 log10TCID50/ml, respectively, and the titer was maintained throughout the study period (Fig. 2). In infected Ae aegypti mosquitoes, CHITV could not be detected in the saliva until the 12th day PI.
Horizontal transmission
Since the Culex mosquitoes demonstrated vector competence, the horizontal transmission studies were only carried out in those two species. Both the species were able to pick up
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CHITV from viraemic mice while feeding, replicate the virus, and transmit it to 2 day old
infant mice, thereby demonstrating horizontal transmission. When used to feed the
mosquitoes, the sick mice (n=8) had virus titers in the range of 5.2 7.0 log10TCID50/ml.
Individual mosquitoes after feeding, had virus titers in the range of 2.3 to 3.2 log10
TCID50/ml. The commencement of sickness in mice bitten by virus exposed Cx
quinquefasciatus and Cx tritaeniorhychus mosquitoes was observed at 48 hr PI, and death
was observed on the 3rd and 4th day PI in mice, respectively (Fig. 3). IFA results showing
positive C6/36 cells confirmed the viral agent responsible for inducing sickness and death in
mice as CHITV (Fig. 4). The virus titers of individual mouse brains of mice infected by virus
exposed Cx quinquefasciatus and Cx tritaeniorhynchus mosquitoes varied from 6.3 to 8.5 and
5.7 to 7 log10TCID50/ml respectively (Fig. 5). The analysis of the survival time showed a
significant difference between the twoDraft species (p=0.03 by the log rank test). The mean
survival times with standard error (SE) for Cx quinquefasciatus and Cx tritaeniorhynchus
were 3.75±0.16 and 4.38±0.18 days, respectively (Fig. 3).
Vertical transmission:
Vertical transmission of CHITV could not be demonstrated in Cx quinquefasciatus and Cx
tritaeniorhynchus mosquitoes in the present study. No change in oviposition and hatching
rates could be observed in the virus exposed mosquitoes having virus titers ranging from 2.3
to 3.2 log10 TCID50/ml. Presence of virus could not be detected in approx 100 F1 generation
mosquitoes of each species screened for CHITV.
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DISCUSSION
The last few decades have witnessed an upsurge in vector borne viral infections with huge impacts on human health (Liang et al. 2015; Mourya and Sudeep 2014). Dengue has become one of the most dreaded arboviral diseases in the world and is endemic in most of the tropical and subtropical countries, with 390 million cases per year (Bhatt et al. 2013). The introduction of West Nile virus into the US, Canada and Europe has been devastating (Barret
2014). Similarly, the re emergence of the chikungunya virus in 2004 in Africa and its subsequent spread to Asia, Europe and the Americas has become a major global concern
(Liang et al. 2015; Petersons and Powers 2016). Zika virus (ZIKV), the recent addition to the long list of emerging/re emerging viruses, has attracted global attention due to severe clinical manifestations viz., Guillain Barre Syndrome in adult patients and microcephaly in children delivered by infected mothers (PlourdeDraft and Bloch 2016).
The re emergence of ZIKV in a virulent form 6 decades after its discovery has suggested the need to characterize widely prevalent innocuous viruses such as CHITV as part of public health preparedness. CHITV, though found circulating in India since 1957, as evidenced by repeated virus isolations and seroprevalence, has not been incriminated as a pathogen of public health importance. Recent studies carried out at the molecular level have shown that a single genotype is circulating in India despite CHITV isolations from different hosts (Yadav et al. 2012). However, there may be a concern, should there be any change in their genome, as BATV has been involved in genetic reassortment with other members of the serogroup. Ngari virus (NRIV), a reassortant formed with S and L RNA segments from the
Bunyamwera virus and the M RNA segment from BATV has been found to be associated with hemorrhagic fever outbreaks in East Africa (Briese et al 2006; Gerrard et al. 2004;
Yanase et al. 2004). Since a number of bunyaviruses are prevalent in India and many of them share the same vectors for transmission, the probability of reassortment is higher.
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In the present study, we have demonstrated CHITV replication, virus dissemination to
legs and saliva in the virus exposed Culex spp. mosquitoes (Fig 2). Culex quinquefasciatus
has a country wide distribution and has been found to be more competent than Cx
tritaeniorhynchus for CHITV transmission. The former had a higher virus titer in the saliva
and induced sickness and death in infant mice faster (Fig. 2). Though no natural isolation of
CHITV has been reported from the mosquito, the high level of replication, the presence of
virus in the saliva and its ability to transmit the virus to susceptible host make it a concerning
potential vector. Cx tritaeniorhynchus mosquitoes were also found to be highly competent to
transmit the virus, which it is not surprising as the virus has been isolated from the species
earlier (Table 1). Since the isolation of the virus is not the only criteria to designate the
mosquito as vector, the present study confirms the role of the mosquito as a vector, as it
replicated and transmitted the virus toDraft a susceptible host. Cx tritaeniorhynchus is abundant in
India and Southeast Asian countries and is the major vector of Japanese encephalitis virus. Cx
gelidus mosquitoes, which yielded the first isolation of BATV in Malaysia, also demonstrated
vector competence for CHITV (Sudeep et al. 2015).
Although Ae aegypti mosquitoes replicated CHITV, the virus could not be detected in
the saliva. This clearly demonstrates that susceptibility and vector competence are different.
In the present study, despite having a body titer of >5 log10 TCID50/ml, the salivary glands
were not found to be infected.
Vertical transmission of CHITV by Cx quinquefasciatus and Cx tritaeniorhynchus
mosquitoes could not be demonstrated in the present study. Since the sample size screened in
the present study is minimal for both the species, it is difficult to rule out this mode of
transmission. More studies with high titer of virus are needed to shed light on this mode of
transmission.
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Most of the initial isolations of CHITV in India were made from Anopheles mosquitoes. However, in the present study, we could not incorporate any data on the vector competence of Anopheles mosquitoes, as our objective was restricted to Aedes and Culex mosquitoes. The potential of the virus to replicate in different mosquito species and humans is a matter of serious concern. The prevalence of the virus in the country, as evidenced by repeated isolations and seroprevalence in vertebrate hosts, the availability of vertebrate hosts for amplification, and the increasing populations of competent mosquitoes pose a serious threat should there be any change in the genome by reassortment or mutation.
Acknowledgements
The authors thank Dr. DT Mourya, Director, NIV for supporting the work with intramural funds; Mr. Atul Walimbe for the statisticalDraft analysis and Mr. RV Aher and Mr. Sachin Dhaigude for technical support. The authors also thank Dr. Atanu Basu and Dr. K Alagarasu for their critical review of the manuscript.
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Legends of Figures:
Fig. 1: Growth kinetics of CHITV in three species of mosquitoes. Experiments were conducted in triplicate with standard error estimates.
Fig. 2: Graph showing virus titers in the legs and saliva of infected Cx quinquefasciatus and
Cx tritaeniorhynchus mosquitoes on different days post infection. Legs and saliva was pooled from 3 5 mosquitoes at each time point so standard error estimates could not be provided.
Fig. 3: Survival curve showing mortality in mice after being bitten by two infected mosquito species Draft Fig. 4: IFA images showing CHITV infected (a) and uninfected (b) C6/36 cells following inoculation with infected mouse brain suspension.
Fig. 5: CHITV titer of individual mouse brains horizontally transmitted by the bite of Cx quinquefasciatus and Cx tritaeniorhynchus mosquitoes
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Table 1: Details of Chittoor virus isolations from India
Strain Year of Place of isolation Host Reference
No. isolation
862711 1986 Kolar, Karnataka Sus scrofa Geevarghese, et al., 1994
804992 1980 Bangalore, Karnataka Culex pseudovishnui NIV unpublished data
804988 1980 Bangalore, Karnataka Culex tritaniorhynchus NIV unpublished data
804986 1980 Bangalore, Karnataka Anopheles subpictus NIV unpublished data
643599-2 1964 Manjari, Pune Culex bitaeniorhynchus Singh &Pavri, 1966
643765 1964 Manjari, Pune Anopheles subpictus Singh &Pavri, 1966
643806-1 1964 Manjari, Pune Anopheles tessellatus, Singh &Pavri, 1966 643806-5 1964 Manjari, Pune DraftAnopheles tessellatus Singh &Pavri, 1966 643946 1964 Manjari, Pune Anopheles tessellatus Singh &Pavri, 1966
G-20217 1957 Chittoor, Andhra Anopheles barbirostris NIV unpublished data
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Table 2: Seroprevalence of CHITV in vertebrates collected from different states of India
Vertebrate host Locations of sera collection %
Seroprevalence
Human Tamil Nadu, Maharashtra, Gujarat 2.2%
Horse Maharashtra, Arunachal Pradesh, Jammu & 62.8%
Kashmir
Goat Tamil Nadu, Maharashtra, Gujarat, Orissa, West 41.8%
Bengal (WB), Uttar Pradesh (UP), Punjab
Sheep Tamil Nadu, Gujarat, WB, UP, Punjab 48.4% Mule Arunachal Pradesh,Draft UP 53.9% Donkey Maharashtra, Gujarat 48%
Camel Gujarat 100%
Cow & Bull Maharashtra, Gujarat, WB, Assam, Arunachal 60.9%
Pradesh
Buffalo Maharashtra, Gujarat, WB 83.3%
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Draft
Fig. 1: Growth kinetics of CHITV in three species of mosquitoes. Experiments were conducted in triplicate with standard error estimates.
254x182mm (300 x 300 DPI)
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Draft Fig. 2: Graph showing virus titers in the legs and saliva of infected Cx quinquefasciatus and Cx tritaeniorhynchus mosquitoes on different days post-infection. Legs and saliva was pooled from 3-5 mosquitoes at each time point so standard error estimates could not be provided.
235x151mm (300 x 300 DPI)
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Draft
Fig. 3: Survival curve showing mortality in mice after being bitten by two infected mosquito species
212x165mm (300 x 300 DPI)
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Fig. 4: IFA images showing CHITV-infected (a) and uninfected (b) C6/36 cells following inoculation with infected mouse brain suspension.
226x89mm (300 x 300 DPI)
Draft
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Draft
Fig. 5: CHITV titer of individual mouse brains horizontally transmitted by the bite of Cx quinquefasciatus and Cx tritaeniorhynchus mosquitoes
217x144mm (300 x 300 DPI)
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