Detection of West Nile virus in six mosquito species in synchrony with seroconversion among sentinel chickens in India
Khan et al. Parasites & Vectors
Detection of West Nile virus in six mosquito species in synchrony with seroconversion among sentinel chickens in India
Siraj A. Khan 0
Purvita Chowdhury 0
Parveena Choudhury 0
Prafulla Dutta 0
0 Regional Medical Research Centre , NE Region, ICMR, Dibrugarh 786001, Assam , India
Background: West Nile virus (WNV) is a zoonotic flavivirus maintained in mosquito-bird transmission cycle. Although humans are accidental hosts, fatal outcomes following WNV infection have been reported from India. Studies have identified WNV as an important etiological agent causing acute encephalitis syndrome in Assam, Northeast India. While circulation of WNV is evident, the role of vectors and avian hosts involved in the transmission remains unclear. In this study we identified local mosquito species for evidence of WNV infection along with seroconversion among sentinel chickens. Methods: Mosquitoes were collected and pooled species wise from June 2014 through December 2015. Virus was screened using reverse transcriptase PCR followed by sequencing and phylogenetic analysis. Sentinel chicken blood was screened for WNV antibody to assess their role in WNV transmission. Results: A total of 52,882 mosquitoes belonging to 16 species were collected. WNV was detected in 18 pools of Culex vishnui, Culex tritaeniorhynchus, Culex quinquefasciatus, Culex whitmorei, Culex pseudovishnui and Mansonia uniformis. Phylogenetic analysis revealed that all mosquito derived sequences belonged to Lineage 5 and were 99-100% similar to the Assam strain of WNV isolated from human CSF sample in 2007. All sentinel chickens had seroconverted by the month of July that happens to be the peak WNV transmission month among humans as well. Conclusion: To the best of our knowledge, this is the first report of WNV identification from field-collected Cx. pseudovishnui and Mansonia uniformis in India. Our study demonstrates potential vectors which may play a crucial role in WNV transmission and should be considered in the vector control strategies. Additionally, our study highlights the role of sentinel chickens for WNV surveillance.
West Nile virus; Lineage 5; Mosquito vectors; Culex pseudovishnui; Mansonia uniformis; Sentinel chickens
West Nile virus (WNV) has emerged as one of the most
widespread flavivirus being reported from all the
continents except Antarctica . Since its first outbreak in
1937, the disease attained immense attention during the
mid-1990s corresponding to its severity, frequency and
geographical expansion [2–4].
WNV is maintained naturally in an enzootic
transmission cycle of mosquitoes (vectors) and birds (amplifying
hosts) while humans, horses and other mammals serve
as accidental/dead end hosts. Among a number of
mosquitoes involved in epizootic and epidemic transmission
including species of Anopheles (in USA, Isreal,
Madagascar), Aedes (in Africa, Russia, USA), Mansonia
(Africa) and Ochlerotatus (USA), the most important
vectors of WNV represent Culex spp. . In India,
WNV has been isolated from Cx. vishnui, Cx.
quinquefasciatus, Cx. tritaeniorhynchus and Cx. fatigans .
House sparrows and corvids have been implicated as
important WNV reservoirs in North America, Europe and
Africa. However, in the Indian subcontinent, ardeid birds
are thought to be the possible amplifying hosts .
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
WNV infection in humans can induce symptoms
ranging from febrile fever to severe neurological syndromes
like encephalitis, meningitis and paralysis . In India,
WNV-neutralizing antibodies were first detected in 1952
. Since then, the virus has been isolated from different
hosts and regions of the country. Recognition of WNV
among acute encephalitis syndrome causing etiologies in
Assam in 2006 was the first report of the flavivirus from
eastern region of India . Genetic characterization of
two WNV isolates obtained from this region revealed
similarity with south Indian WNV Lineage 5 strains .
Although WNV circulation in this region is evident,
adequate knowledge of vectors and amplifying hosts
involved in transmission of WNV are lacking. In this study
we investigated local mosquito species as candidate WNV
vectors along with the role of birds as amplifying hosts.
Study sites were selected based on maximum number and
frequency of WN cases reported during earlier outbreaks in
the eastern regions of the State of Assam. Adult mosquitoes
were collected from four townships: Dibrugarh (27.4728°N,
94.9120°E); Tinsukia (27.4922°N, 95.3468°E); Sivasagar
(26.9826°N, 94.6425°E); and Duliajan (27.3572°N, 95.3223°
E) (Fig. 1). The sites were kept unchanged throughout the
study. The selected townships have numerous water bodies
that serve as abodes for migratory birds during winters.
Mosquito collection was carried out for 1–2 h during
dusk using mechanical aspirators at fortnightly intervals
from June 2014 through December 2015. Mostly cattle
sheds were targeted which are open on three sides with
a close proximity to human dwellings. All mosquitoes
were identified following standard entomological keys
[12–14] under immobilization (by cold shock) and
pooled species wise for incrimination studies. Although
we did not calculate the proportionate ratio, it
comprised of non-fed, partially-fed as well as fully-fed
mosquitoes. However, the fully-fed ones were kept in the
insectarium for over 24 h for digestion of the blood meal
before identification and pooling. Species wise mosquito
man hour density (MHD) was calculated as
MHD = number of mosquitoes collected × 60/time spent
in minutes × number of persons involved in collections.
Every mosquito pool was triturated mechanically using 2%
foetal bovine serum (FBS) (Gibco, Thermo Fisher
Scientific, Massachusetts, USA) in pre chilled minimum
essential medium (MEM) (Sigma-Aldrich, St. Louis, MO, USA)
treated with 50 U penicillin (Sigma-Aldrich, St. Louis,
MO, USA), 50 μg/ml streptomycin (Sigma-Aldrich, St.
Louis, MO, USA), and 50 μg/ml amphotericin B
(SigmaAldrich, St. Louis, MO, USA). The homogenate after
centrifugation was processed for RNA extraction as per
manufacturer’s instructions (QIAamp Viral RNA Kit,
Presence of WNV RNA was screened in all the
mosquito pools using a semi-nested reverse transcriptase
(RT)-PCR performed on verity 96 well thermal cycler
(Applied Biosystems, California, USA). Primers used for
WNV RNA amplification have been described by Khan
et al.  giving a 500 bp product. Briefly, the first
round of amplification was performed with 2 μl of
Fig. 1 Locations of mosquito collection in Assam, India. Mosquitoes were collected fortnightly at the four sites from June 2014 through December 2015
suspended RNA template using Access one step RT-PCR
kit (Promega, Wisconsin, USA). The first PCR amplicon
(1 μl) was then subjected to semi nested PCR using 2×
Master Mix (Promega, Wisconsin, USA) with 200nM of
reverse primer (sn5'-TGG CCA AGA ACA CGA CCA
GAA GG-3') in a final volume of 15 μl. PCR profile for
the second round of amplification were carried out by
denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C
for 30 s, 54 °C for 1 min and 72 °C for 1 min and final
extension at 72 °C for 7 min. All positive amplicons were
confirmed by sequencing commercially (Avantor, Selangor,
The resulting forward and reverse sequences were
manually edited in BioEdit version 7.0.9 software .
The edited nucleotide sequences were compared with a
total of 20 WNV GenBank sequences which were
selected on the basis of their lineage and geographical
origin for determination of WNV lineage. A GenBank
sequence (AF080251) of Indian Japanese encephalitis
virus (JEV) strain was used as the out-group.
Phylogenetic analysis was carried out using Mega 7 software .
The Clustal W program implemented in Mega 7
software was used to generate a multiple alignment of the
sequences and subsequently construct character based
maximum likelihood (ML) tree. Nucleotide and amino
acid sequence similarity was estimated in Mega 7 using
the gamma distribution (shape parameter = 5). Reliability
of the tree was estimated by 1000 bootstrap replications.
In cooperation with the Forest Department, Government
of Assam, India, a strict vigilance on any unusual
mortality of birds in the reserve forests and national parks of
Assam was maintained. However, no such incidence
(either among migratory or local birds) was reported
during the study period. Subsequently, we attempted to
look for WNV seroconversion in sentinel chickens to
ascertain the role of domestic birds in WNV transmission
as circulation of WNV has been evident among humans
in this region for the past decade . Sentinel chickens
with each flock consisting of 10 birds were established at
two of the study sites, Dibrugarh and Sivasagar, during
April 2015 - August 2015. Blood samples were collected
fortnightly by using brachial venipuncture method, and
the separated serum was stored at -80 °C until
serological tests were performed.
To detect WNV-specific antibodies, chicken serum
samples were tested in 96 well microtitre plates using
hemagglutination inhibition (HI) test . Briefly,
removal of nonspecific inhibitors in chicken sera was
achieved by acetone extraction. Acetone treated chicken
sera were serially diluted and mixed with four
hemagglutination (HA) units of WNV antigen. Following
an overnight incubation at 4 °C, goose red blood
corpuscles (RBCs) were added and the solution was incubated
at room temperature for another hour. The HI titre was
expressed as the highest serum dilution producing
complete inhibition of RBC agglutination. Sera with HI
titre of 1:10 were considered as positive. Due to
crossreactivity among JEV and WNV in endemic region, the
chicken sera were also tested against JEV antigen.
Consequently, WNV antibodies were distinguished by
comparing both WNV and JEV HI titres .
The minimum infection rate (MIR) was calculated as the
number of infected mosquitoes per 1000 mosquitoes
tested . Differences in chicken seroprevalence rates
among the two study sites were tested by unpaired
Student’s t-test. The temporal concordance of sentinel
chicken seroconversion and mosquito MHD was
evaluated by cross correlation analysis.
Mosquito samples and viral detection
During June 2014 - December 2015, a total of 52,882
adult mosquitoes belonging to 16 species (including 10
known potential vectors of WNV) were collected and
analysed. Our collections showed an almost equal
distribution of mosquito density in all the study sites and the
total number of samples per site is given in Table 1.
Mansonia uniformis, Culex vishnui and Culex
tritaeniorhynchus were the predominant species constituting
26.32, 24.47 and 13.50% of the total catch, respectively.
Eighteen of the 1392 mosquito pools tested RT-PCR
positive for WNV non-structural 1 (NS1) gene. Majority
of the positive pools consisted of Ma. uniformis and Cx.
vishnui followed by Cx. tritaeniorhynchus, Cx.
quinquefasciatus, Cx. pseudovishnui and Cx. whitmorei (Table 2).
The MIR of Cx. whitmorei was found to be highest.
Partial sequence of WNV NS1 region of .500 bp was
obtained from 18 mosquito pools (GenBank Accession
nos. KX646169–KX646186). The derived phylogenetic tree
revealed that the sequences obtained during the study
formed a clade within the Lineage 5 in the tree (Fig. 2).
Furthermore, it indicated that the circulating WNV in Assam
during 2014–2015 was similar to the Lineage 5 strain
isolated in Assam during 2007 (HQ246154). An additional file
shows that all the WNV positive sequences were more than
99% similar to the 2007 Assam isolate (see Additional file 1:
Table S1). The mean genetic distance of 0.01% was found
at the nucleotide level. On alignment of each
aPotential WNV vector
derived sequence with 2005 Indian strain (DQ256376) and
2007 Assam strain (HQ246154), a few synonymous and
non-synonymous mutations were observed. Interestingly,
two synonymous mutations, i.e. C → T and T → G, were
found in all mosquito sequences except for KX646169.
Moreover, two nucleotide substitutions resulted in changes
in amino acid codons, i.e. from valine to alanine in three
mosquito-derived sequences. It is worth mentioning that
one of the sequences (KX646186) formed a distinct
subclade pertaining to a few synonymous and
It was observed that 70% of the chicken sera had higher
WNV compared to JEV HI titer. Seroconversion in
sentinel chickens started towards end of May and antibodies
against WNV were detected in all sentinel chicken by
Table 1 Distribution of adult mosquitoes of sixteen species
collected at the four study sites
July 2015. Significant differences in seroprevalence rates
among the two study sites were not observed in the
present study. The percentage of chicken seroconversion
during May to August 2015 was compared with the
abundance of incriminated WNV vectors. Chicken
seroconversion peak coincided with the increase in
abundance of three incriminated WNV vectors, Ma.
uniformis, Cx. vishnui and Cx. whitmorei. However,
peaks in abundance of Cx. pseudovishnui and Cx.
tritaeniorhynchus were preceded by peaks in chicken
seroconversion. The cross correlation with 95% CI revealed that
sentinel chicken seroconversion (r = 0.77, P = 0.06) was
marginally associated to the density of Ma. uniformis
mosquito after 2 weeks (Fig. 3).
The present study detected WNV belonging to Lineage
5 from six species of mosquitoes and corroborated that
the transmission of WNV involves bird-mosquito cycle
in upper basins of the Brahmaputra valley of Assam,
Northeast India. The geographical distribution and
abundance of potential WNV vectors at all of the four
study sites suggests the possibility of active circulation of
WNV. Cattle sheds targeted during mosquito collection
harbor abundant zoophilic mosquito species. As almost
all the potential/incriminated vector species are
exophilic and exophagic in nature, it is unlikely that important
mosquito species could have been missed. As
anticipated, Ma. uniformis and Cx. vishnui subgroup were
highly abundant in the study areas similar to previous
study conducted by Khan et al. . These species
happen to be established JEV vectors as well, which may
explain the concurrent outbreaks of WNV and JEV in this
region. An earlier study has also evidenced coinfection
of both WNV and JEV in humans from this region
during outbreaks in 2007 .
As sentinel chickens do not produce transmissible
viremia, molecular analysis was not performed for the
sampled chicken sera. However, molecular analysis of
mosquito pools revealed WNV in Ma. uniformis, Cx.
vishnui, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx.
pseudovishnui and Cx. whitmorei. The role of Culex
Table 2 Mosquito species found to be WNV-positive by RT-PCR and sequencing from the four study sites
Abbreviation: MIR minimum infection rate; calculated as (number of positive pools/total number of specimen tested) × 1000
Fig. 2 Phylogenetic analysis of geographically distinct West Nile virus sequences based on 500 bp region of the NS1 gene. Black triangles denote
the mosquito-derived sequences from this study. The maximum likelihood tree was constructed using Kimura 2-parameter model in Mega 7. An
Indian strain (P-20778) of Japanese encephalitis virus (GenBank accession no. AF080251) was used as an outgroup. Node values were estimated
for 1000 replicates
species, especially Cx. quinquefasciatus have been well
documented and implicated in WNV transmission in
North America, Europe, South Africa and Australia .
In India, WNV has been isolated from Cx. vishnui during
1955–1958 and 1980–1981, and from Cx. whitmorei and
Cx. tritaeniorhynchus in 1980. Mansonia uniformis have
been found to carry WNV in Madagascar and Ethiopia
[23, 24]. However, our study describes the first report of
Ma. uniformis as a potential WNV vector in India. On the
other hand, field-collected Cx. pseudovishnui has never
been incriminated for WNV infection, although, a few
experimental studies implied it to be a candidate WNV
vector [25, 26]. Our study provides evidence that Cx.
pseudovishnui is a potential vector for WNV transmission
in this region. The incriminated mosquito species in our
study are known as zoo-anthropogenic [27, 28]. Blood
meal analysis of Cx. quinquefasciatus has demonstrated it
to be highly ornithophilic besides feeding on mammals
. It is most likely that that this vector acts as an
important bridge vector and aid in viral amplification.
Fig. 3 Relationship between Ma. uniformis MHD and lagged sentinel chicken seroconversion (lead and lag shown on the x-axis) represented by
correlation coefficient with 95% confidence interval
Phylogenetic analysis revealed that all the sequences
belonging to WNV Lineage 5 are similar to earlier
report of WNV from Assam, India . Based on isolates
obtained from mosquitoes and humans in previous
studies, there has been a prominent circulation of WNV
Lineage 5 in India since its first detection in 1955 .
In our study, a distinct clade formed between the
sequences from mosquitoes (2014–2015) and a WNV
isolate from humans (2007) in Assam. This indicates a
local circulation of this particular strain of Lineage 5
WNV in the given situation. However, the implications
of synonymous and non-synonymous mutations
observed in WNV strain in mosquito species require
The hemagglutination inhibition (HI) test used in this
study has been considered as one of the standards for
measuring neutralizing antibody titre. Previous studies
have demonstrated the test to be specific as well as
sensitive which is comparable to micro virus neutralization test
. Serological evidence of WNV infection in wild
resident birds has been previously observed in Northern and
Eastern India . In this study, WNV antibodies were
observed in sentinel chickens during the monsoon season:
May-July, which is conducive for increase of potential
vectors in this region. Although seroconverted chickens were
not replaced with naïve ones and a low number of sentinel
chicken were considered in this study, it indicated local
WNV transmission that may be employed as an indicator
of WNV activity in this region. The use of sentinel chicken
to monitor WNV activity has been widely incorporated in
many countries with WNV transmission including USA
. However, the involvement of other domestic birds of
this region needs to be further investigated as earlier
reports have shown WNV seroprevalence in domestic ducks
and turkey [24, 34]. Wild migratory birds are often seen in
this region, visiting water bodies and the banks of
Brahmaputra, which is 10–15 km from all the study sites.
Future studies involving sampling from migratory and other
domestic birds may elucidate their role in the WNV
transmission in this region.
Our study found two new potential WNV vectors that
could be playing a crucial role in WNV transmission in
India. Detailed virus transmission studies as well as
blood meal analysis of incriminated mosquito vectors
would provide more evidence of the WNV cycle.
Additionally, we corroborated that sentinel chickens can be
useful to monitor WNV activity. These findings expand
our understanding on vector diversity of WNV and help
expedite future studies on the eco-epidemiology of
Additional file 1: Table S1. Nucleotide divergence among WNV
Lineage 5 sequences. (XLSX 10 kb)
HA: Hemagglutination; HI: Hemagglutination inhibition; JEV: Japanese
encephalitis virus; ML: Maximum likelihood; NS1: Non-structural 1; RBC: Red
blood corpuscles; RT-PCR: Reverse transcriptase polymerase chain reaction;
WNV: West Nile virus
The authors thank the entire arbovirology division of Regional Medical
Research Centre, NE Region, Dibrugarh. The authors are also grateful to
Nibedita Das, Dipjyoti Baruah, Pranab Saikia and Pranjal Mahanta for their
excellent technical support.
This study was supported by Indian Council of Medical Research, New Delhi,
India. The funding agency did not have any role in study design, collection,
analysis and preparation of the manuscript.
Availability of data and materials
All data generated or analysed during the current study are included in this
article and its Additional file. The sequences are submitted in the GenBank
database under accession numbers KX646169–KX646186.
SAK and PD designed and planned the study. Purvita Chowdhury and
Parveena Choudhury performed the laboratory work and analyzed the data.
SAK and Purvita Chowdhury drafted the manuscript. All authors read and
approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The study was approved by the Institutional Ethics Committee of Regional
Medical Research Centre, ICMR, Dibrugarh, Assam, India and sampling was
carried out by trained personnel.
1. HayesCG . West Nile fever . In: Monath TP, editor. The arboviruses: epidemiology and ecology , Vol. V. Boca Raton (FL): CRC Press; 1989 . p. 59 - 88 .
2. Smithburn KC , Hughes TP , Burke AW , Paul JH. A neurotropic virus isolated from the blood of a native of Uganda . Am J Trop Med . 1940 ; 20 : 471 - 92 .
3. Tsai TF , Popovici F , Cernescu C , Campbell GL , Nedelcu NI . West Nile encephalitis epidemic in southeastern Romania . Lancet. 1998 ; 352 ( 9130 ): 767 - 71 .
4. Weinberger M , Pitlik SD , Gandacu D , Lang R , Nassar F , David DB , et al. West Nile fever outbreak, Israel , 2000 : epidemiologic aspects. Emerg Infect Diseases . 2001 ; 7 ( 4 ): 686 .
5. Wildpro-the electronic encyclopaedia and library for wildlife . Wildlife Information Network , Warwickshire, UK. 2013 . http://wildpro.twycrosszoo. org/S/virus/flaviviridae/Flaviviridae_WNVirus/06WNVSppIntermediateHost. htm. Accessed 11 Dec 2015 .
6. Paramasivan R , Mishra AC , Mourya DT . West Nile virus: the Indian scenario . Indian J Med Res . 2003 ; 118 : 101 - 8 .
7. Rodrigues FM , Guttikar SN , Pinto BD . Prevalence of antibodies to Japanese encephalitis and West Nile viruses among wild birds in the Krishna-Godavari Delta , Andhra Pradesh , India. Trans R Soc Trop Med Hyg . 1981 ; 75 ( 2 ): 258 - 62 .
8. Komar N. West Nile viral encephalitis . Revue scientifique et technique (International Office of Epizootics) . 2000 ; 19 ( 1 ): 166 - 76 .
9. Banker DD . Preliminary observations on antibody patterns against certain viruses among inhabitants of Bombay city . Indian J Med Sci . 1952 ; 6 : 733 - 46 .
10. Khan SA , Dutta P , Khan AM , Chowdhury P , Borah J , Doloi P , Mahanta J. West Nile virus infection , Assam, India. Emerg Infect Diseases . 2011 ; 17 ( 5 ): 946 - 8 .
11. Chowdhury P , Khan SA , Dutta P , Topno R , Mahanta J. Characterization of West Nile virus (WNV) isolates from Assam, India: insights into the circulating WNV in northeastern India . Comp Immunol Microbiol Infect Dis . 2014 ; 37 ( 1 ): 39 - 47 .
12. Barraud PJ . The fauna of British India , including Ceylon and Burma. Diptera. Volume V. Family Culicidae. Tribes Megarhinini and Culicini. London: Taylor and Francis ; 1934 .
13. Nagpal BN , Sharma VP . Survey of mosquito fauna of northeastern region of India . Indian J Malariol . 1987 ; 24 : 143 - 9 .
14. Christopher SR . The fauna of British India, including Ceylon and Burma, Diptera, Volume IV. New Delhi: Today and Tomorrow 's Printers and publishers; 1933 .
15. Khan SA , Dutta P , Chowdhury P , Borah J , Topno R , Mahanta J. Coinfection of arboviruses presenting as Acute Encephalitis Syndrome . J Clin Virol . 2011 ; 51 ( 1 ): 5 - 7 .
16. Hall T . BioEdit version 7 .0. 0. Distributed by the author . 2004 . website: www . mbio.ncsu.edu/BioEdit/bioedit.html. Accessed 20 Dec 2013 .
17. Kumar S , Stecher G , Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets . Mol Biol Evol . 2016 :msw054. doi:10. 1093/molbev/msw054.
18. Clarke DH , Casals J. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses . Am J Trop Med Hyg . 1958 ; 7 ( 5 ): 561 - 73 .
19. Hirota J , Shimizu S , Shibahara T , Kobayashi S. Cross-reactivity of chicken antiJapanese encephalitis virus serum and anti-West Nile virus serum in serological diagnosis . J Vet Med Sci . 2012 ; 74 ( 11 ): 1497 - 9 .
20. Chiang CL , Reeves WC . Statistical estimation of virus infection rates in mosquito vector populations . Am J Trop Med Hyg . 1962 ; 75 : 377 .
21. Khan SA , Narain K , Handique R , Dutta P , Mahanta J , Satyanarayana K , Srivastava VK . Role of some environmental factors in modulating seasonal abundance of potential Japanese encephalitis vectors in Assam , India. Southeast Asian J Trop Med Public Health . 1996 ; 27 : 382 - 91 .
22. Chancey C , Grinev A , Volkova E , Rios M. The global ecology and epidemiology of West Nile virus . BioMed Res. Int . 2015 . http://dx.doi.org/10. 1155/2015/376230. Accessed 16 Aug 2016 .
23. Peiris JSM , Amerasinghe FP . West Nile fever . In: Beran GW, Steele JH, editors. Handbook of zoonoses. Section B: Viral . 2nd ed. Boca Raton (FL): CRC Press; 1994 . p. 139 - 48 .
24. Maquart M , Boyer S , Rakotoharinome VM , Ravaomanana J , Tantely ML , Heraud JM , Cardinale E. High prevalence of West Nile virus in domestic birds and detection in two new mosquito species in Madagascar . PLoS One . 2016 ; 11 ( 1 ): e0147589 .
25. Akhter R , Hayes CG , Baqar S , Reisen WK. West Nile virus in Pakistan . III. Comparative vector capability of Culex tritaeniorhynchus and eight other species of mosquitoes . Trans R Soc Trop Med Hyg . 1982 ; 76 ( 4 ): 449 - 53 .
26. Mishra AC , Jadi RS , Paramasivan R , Mourya DT . Antigen distribution pattern of West Nile virus in Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes . J Comm Dis . 2001 ; 33 ( 3 ): 174 - 9 .
27. Reuben R , Thenmozhi V , Samuel PP , Gajanana A , Mani TR . Mosquito blood feeding patterns as a factor in the epidemiology of Japanese encephalitis in southern India . Am J Trop Med Hyg . 1992 ; 46 ( 6 ): 654 - 63 .
28. Bhattacharyya DR , Handique R , Dutta LP , Dutta P , Doloi P , Goswami BK , et al. Host feeding patterns of Culex vishnui sub group of mosquitoes in Dibrugarh district of Assam . J Comm Dis . 1994 ; 26 ( 3 ): 133 - 8 .
29. Garcia-Rejon JE , Blitvich BJ , Farfan-Ale JA , Loroño-Pino MA , Chim WC , FloresFlores LF , et al. Host-feeding preference of the mosquito, Culex quinquefasciatus , in Yucatan State, Mexico . J Insect Sci . 2010 ; 10 ( 32 ): 1 - 2 .
30. Bondre VP , Jadi RS , Mishra AC , Yergolkar PN , Arankalle VA . West Nile virus isolates from India: evidence for a distinct genetic lineage . J Gen Virol . 2007 ; 88 ( 3 ): 875 - 84 .
31. Weingartl HM , Drebot MA , Hubálek Z , Halouzka J , Andonova M , Dibernardo A , et al. Comparison of assays for the detection of West Nile virus antibodies in chicken serum . Can J Vet Res . 2003 ; 67 ( 2 ): 128 .
32. Mishra N , Kalaiyarasu S , Nagarajan S , Rao MV , George A , Sridevi R , et al. Serological evidence of West Nile virus infection in wild migratory and resident water birds in eastern and northern India . Comp Immunol Microbiol Infect Dis . 2012 ; 35 ( 6 ): 591 - 8 .
33. Fall AG , Diaïté A , Seck MT , Bouyer J , Lefrançois T , Vachiéry N , et al. West Nile virus transmission in sentinel chickens and potential mosquito vectors , Senegal river delta , 2008 - 2009 . Int J Environ Res Publ Health . 2013 ; 10 ( 10 ): 4718 - 27 .
34. Ergunay K , Gunay F , Kasap OE , Oter K , Gargari S , Karaoglu T , et al. Serological , molecular and entomological surveillance demonstrates widespread circulation of West Nile virus in Turkey . PLoS Negl Trop Dis . 2014 ; 8 ( 7 ): e3028 .