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Multiple dengue virus types harbored by individual mosquitoes
Accepted Manuscript Title: Multiple dengue virus types harboured by individual mosquitoes Author: Bennet Angel Annette Angel Vinod Joshi PII: DOI: Reference:
S0001-706X(15)30056-5 http://dx.doi.org/doi:10.1016/j.actatropica.2015.07.007 ACTROP 3675
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
14-1-2015 2-7-2015 9-7-2015
Please cite this article as: Angel, Bennet, Angel, Annette, Joshi, Vinod, Multiple dengue virus types harboured by individual mosquitoes.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multiple dengue virus types harboured by individual mosquitoes
Bennet Angel, Annette Angel and Vinod Joshi* Laboratory of Virology & Molecular Biology, Desert Medicine Research Centre, Indian Council of Medical Research, Jodhpur, India *Corresponding author: Dr. Vinod Joshi, Laboratory of virology & Molecular Biology, Desert Medicine Research Centre, Pali Road, Jodhpur-342 005, India. Telephone: 0912912729729; 09660767977; Fax: 0912912720618 E-mail: [email protected] Highlights
Multiple DENV types are present in individual mosquitoes from Rajasthan, India
The mosquitoes could be non human source of hetero DENV types in endemic settings.
Paper sensitizes further studies on multiple DENV types in mosquitoes and DHF.
Abstract The existing knowledge on pathogenesis and aetiology of DHF establishes that Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) are caused by two subsequent infections of two different serotypes of dengue affecting a common human population with a time gap. Present studies have been undertaken on 212 laboratory reared infected individual mosquitoes from larvae collected from 31 dengue endemic towns of Rajasthan, India. Type specific DEN viruses were detected from individual mosquitoes employing RT-PCR. In 78.7% of 212 infected individual mosquitoes studied, vertically transmitted multiple DENV types were observed. We report for the first time that single mosquitoes contain multiple dengue virus types. Keywords: Multiple dengue types; Etiology; DHF
Dengue Fever (DF) with Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) is an emerging public health problem with an estimated number of 390 million cases reported annually (Bhatt and Brady, 2013). The severe form of dengue ie, DHF or DSS, responsible for case fatality up to more than 5% (Gubler, 2011) is believed to be associated with an Antibody Dependent Enhancement (ADE) in the patients (Halstead and Rourke, 1977), possibly caused by infectious immune complexes formed between a current DENV infection and non neutralizing antibodies already present in the patient’s system (Guzman et al., 2010). As of today we stand with an understanding that multiple infections of more than one DENV types with a time gap is a pre-requisite for DHF. To understand infection of different DENV types in a population with a gap of time, more emphasis has been given so far on isolation of DENV types (DEN-1, 2, 3 or 4) from the human patients (Guzman, 1981). Although dengue transmission involves both, horizontal as well as vertical transmission of virus (WHO, 2009), latter has not been given adequate importance. Mosquitoes, in addition to transmit virus from man to man, may also contribute extrinsic or unchallenged viral stock passaged by its own system by virtue of transovarial transmission of virus (Khin and Than, 1983; Rosen and Gubler, 1974; Joshi etal., 2002). We hypothesized that studies to comprehend the DENV types contained by single mosquito in a setting could add to our knowledge on non human source of multiple dengue types in an endemic area. During the course of present investigations we have undertaken molecular isolation of dengue virus types from individual specimens of laboratory reared Aedes aegypti from larvae collected from 31 disease endemic districts towns of Rajasthan state, India. The paper reports first observations on presence of multiple dengue virus types carried by individual
mosquitoes and discusses the possible significance of the observations in aetiology of DHF.
Studies were undertaken in all 33 district headquarter towns of north-western state Rajasthan, India. Rajasthan represents the arid ecosystem forming western boarder of India. It includes 33 districts which form a north-western desert part, an eastern semiarid part and southern hilly area. Out of 33 district headquarter towns, 28 are endemic for dengue fever (Unpublished data, state health department, Government of Rajasthan, India). The areas from where larval mosquitoes were collected are 31 district headquarter towns as listed at the foot note (Table). No breeding was seen in two districts i.e. Udaipur and Dholpur. Overall a total of 19,775 households were surveyed.
Larvae of Aedes aegypti were collected from the domestic and peri-domestic water collections from different localities in the urban settings of 31 dengue endemic districts of state of Rajasthan, India from May, 2012 till May 2014 using random sampling design. Random numbers were assigned to selected houses in study towns. Depending upon the size of town, every 6th, 8th or 10th house was chosen. All the breeding containers available in a sampled house were examined for larval collection. A total of 1, 30, 525 containers were surveyed. The larvae were brought and reared in laboratory into adult mosquitoes at ambient temperature of about 25˚C. Larval food of dog biscuit and yeast powder was provided. Adult Aedes aegypti emerged were kept in Barraud cages maintained individually for each locality in each district for about 56 days at about 25˚C and relative humidity of 65%.
Individual Aedes adults were homogenized in 200µl PBS and centrifuged at 5000 rpm for 10 minutes at 4˚C. The supernatant were transferred to clean eppendorfs, labelled district wise and stored at -80˚C. For screening the presence or absence of dengue virus antigen within them, 5µl of this supernatant (before storing) was subjected to Indirect Fluorescence Antibody test (IFAT) and a green fluorescence observed was recorded as IFAT positive result.
Thus a total number of 5136 laboratory reared mosquitoes were screened employing Indirect Fluorescence Antibody Test (IFAT), of which 1263 (24.59%) were found positive by dengue virus. Of these 1263 vertically infected mosquitoes, 212 specimens were selected (6-10 mosquitoes from each setting) and subjected to DENV type virus detection employing RT-PCR.
Viral RNA was extracted using the QIAmp Viral RNA kit (m/s Qiagen Inc., Valencia, CA). Gene specific primers (Lanciotti et al, 1992) were used for detecting the four types of DEN virus in the individual samples. These primers used were commercially synthesized from m/s Eurofins, Bangalore, India. Thus for each sample a total of 4 reaction was run, one representing a single type. The Superscript III RT/Taq Polymerase One-Step RT-PCR kit (manufactured by m/s Invitrogen, USA) was used for the assay following the manufacturer’s protocol. The temperature conditions were also as suggested in the protocol. After the amplification, 10µl of PCR product (four for each sample) was run on a 2% Agarose gel (m/s Invitrogen, USA) and then viewed in the Gel Documentation System (manufactured by m/s Bio-Rad, USA). This was done for a total of 212 individual mosquitoes. The types that appeared as bands were noted for each sample. Of these, eight mosquitoes were selected for sequencing.
Besides these 8 mosquitoes, viral suspensions of 6 infected mosquitoes were inoculated in Cell lines of C6/36 (Aedes albopictus) procured commercially from National Centre for Cell Sciences (NCCS), Pune, India. 420 µl of this suspension was subjected to RNA extraction and RT-PCR amplification as mentioned above. The primers used were as that referred in Lanciotti et al, 1992. These cell-line inoculated mosquito samples were also reacted with another set of type-specific primers (Seah et al, 1995). After detecting the type-specific bands for each sample in the Agarose gel, the remaining sample was purified using the PCR purification kit (manufactured by m/s Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The final purified product was eluted in 10 µl of Elution buffer provided in the kit. Thus a total of 14 samples of infected mosquitoes showing multiple DEN-V types were subjected to sequencing.1-2 µl of this PCR purified product was subjected to cycle sequencing using the Big Dye Terminator cycle sequencing kit v3.1 (manufactured by m/s Invitrogen, USA). This was followed by purification of the cycle sequenced product using the Dye-Ex 2.0 spin kit (manufactured by m/s Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The purified product was then loaded in the 16-capillary based 3130XL Genetic Analyzer (m/s ABI, USA).
Data generated from the sequencer were then analyzed using the ‘Sequence Analysis’ software. The quality check and trimming of the sequences generated was done using the ‘Seqscape 2.6’ software (m/s Applied Biosystems, USA) and then attempted for their assembly with the available dengue virus type-specific reference genomes. After trimming, BLAST was carried out. Further, multiple alignment was also performed using the ‘Clustal-W’ for the samples by placing them in four categories according to their types identified (Den-1, 2, 3 or 4).
Hence in all, a total number of 1, 30, 525 domestic water containers from 31 district towns of Rajasthan, India were searched for larval collection of Aedes mosquitoes. Total number of 5136 laboratory reared mosquitoes were screened for IFAT of which 1263 (24.59%) were found positive by vertically transmitted dengue virus. Of these, 212 (174 females and 38 males) specimens were selected from 31 study settings (610 mosquitoes from each district) for type-specific RT-PCR. Table shows results of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for DENV types as observed in individual mosquitoes. It was observed that in 78.7% of total specimens studied, more than one DEN type was present. Further analysis of data of mixed infections showed that in 163 out of 212 mosquitoes (76.8%) DEN-2 was observed, in 155 (73.1%) DEN-3 was present, in 132 (62.2%) DEN-4 was present and least 53 (25%) specimens DEN-1 was observed. The sequences of the 14 samples (processed for sequencing), obtained after trimming when assembled and annotated with the reference
genomes
of
the
four
genotypes
(NC_001477.1,
NC_001474.2,
NC_001475.2, NC_002640.1 respectively) gave no match. But when these sequences were matched and aligned with each other using Clustal-W software showed good coverage and identity. The results of this multiple sequence alignment showed presence of multiple DEN-V types in 6 of 14 mosquito samples while rest showed single or no DEN-V types. Of these, one mosquito sample showed good coverage for all the four DEN-V types, 5 showed good coverage for two DEN-V types and five showed coverage for single DEN-V type. The sequencing results of the six mosquitoes inoculated in cell lines and amplified with second set of primers (Seah et al, 1995) showed good assembly and annotation with DENV-2. The sequences generated will be attempted for submission in the NCBI website.
It is believed that primary infection by any of the serotypes of dengue leads to simple dengue fever (Guzman et al., 2010), whereas infection by two different dengue strains with a time interval may lead to DHF/DSS (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000). Experimental basis of above hypothesis is the DENV type specific virus isolations from human population cohort exhibiting DHF/DSS. In addition, the observations on experimental infection of monkeys by different DENV types with time intervals have also contributed to strengthen above hypothesis (Halstead et al., 1973). However, limitations of the above studies (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000) is that DENV types have been observed from serum samples of cohort of human patients which represents an immunologically challenged virus stock. Limitations of the other studies in supporting above hypothesis is that earlier workers (Halstead et al., 1973), have performed experiments on 118 rhesus monkeys but only one animal has manifested signs of DHF viz; thrombocytopenia and elevation of prothrombin, when challenged by different DENV types with a time interval. The aetiology and pathogenesis of DHF/DSS are still not fully understood (Gubler, 1998). Although earlier workers (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000) have reported enough serological evidences to explain pathogenesis of DHF, however none of the studies has included study of DENV types from mosquitoes. It is well known that dengue viruses undergo transovarial transmission (Khin and Khin, 1983; Rosen and Gubler, 1974). Experimental studies have also shown persistence of transovarial transmission in successive generations of infected mosquitoes (Joshi et al., 2002; Halstead et al., 1973; Shroyer, 1990).
We
hypothesized that transovarial transmission of dengue virus, in addition to serve as the possible mechanism of virus retention in nature (Joshi et al., 2002), may also conserve the hetero DENV types in study settings. Based on our observations of detection of
DENV types from 212 individual infected mosquitoes from 31 disease endemic settings of Rajasthan, India of which 78.7% carried more than one DENV type. We report for the first time that vertically infected individual mosquitoes possess multiple DNV types.
Present observations highlight two distinct messages. One is that in dengue endemic settings, through transovarial route, multiple DENV types establish themselves across mosquito generations. Since we have made RT-PCR virus isolations on individual mosquitoes and not on the pools of mosquitoes, these observations carry their own significance to think whether repetitive bites of mosquitoes infected by multiple DENV types could serve to provide source of hetero DENV types
in endemic
settings. Present observations get supported by the earlier reports citing that among young children even primary infection of dengue leads to vascular leakage and that age dependant host factors contribute to such manifestations (Alvarez, 2006; Guzman, 2002; Gamble 2000).
Acknowledgements We thank Director, Desert Medicine Research Centre (Indian Council of Medical Research), Jodhpur for providing facilities and support in conducting above work. Present work was supported by the Indian Council of Medical Research, Ministry of Health & Family Welfare, Government of India, India. We also thank Sh. Ajay Prakash Joshi, Scientist-I, Dr. Rajendra Kumar Baharia, Scientist-II and Smt. Suman Rathore, Research Assistant, Desert Medicine Research Centre, Jodhpur for providing support for Bioinformatics analysis.
References Alvarez, 2006. Alvarez M. Dengue hemorrhagic fever caused by sequential dengue 13 virus infections over a long time interval: Havana epidemic 2001-2002. Am J Trop Med Hyg 75 (2006), pp. 1113-1117. Bhatt et al, 2013. Bhatt, S. and P. W. Gething, O. J. Brady. The global distribution and burden of dengue. Nature 496 (2013), pp. 504-507. Gubler, 1998. Gubler, D. J. Dengue and Dengue Hemorrhagic Fever. Cli Micro Rev 11(1998), pp. 480-496. Gamble, 2000. Gamble, J. Age related changes in microvascular permeability: a significant factor in susceptibility of children to shock. 2000. Clin Sci 98 (2000), pp.211-216. Guzman, 2002. Guzman, M.G. Enhanced severity of secondary dengue-2 infections. Death rates in 1981 and 1997 Cuban outbreaks. Rev Panam Salud Publica 11 (2002), pp. 223-227. Guzman et al., 2010. Guzman, M. G., Halstead, S. B, Artsob, H., Buchy, P. Dengue: A continuing global threat. Nature: (2010), pp. 7-16. Gubler, 2011. Gubler, D. J. Dengue, Urbanization and globalization: The unholy treaty of 21st century. Trop Med Hlth 39 (2011), pp. 3-11. Guzman, 1990. Guzman, M. G. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seo epidemiologic study. 1990. Am J Trop Med Hyg 42 (1990), pp. 179184. Halstead and O’Rourke, 1977. Halstead, S. B., O’Rourke, E. J. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non neutralizing antibody. J Exp med 146 (1977), pp. 201-217. Halstead et al, 1973. Halstead, S.B., Henry, S., Jordi, C. Studies on the Pathogenesis of Dengue Infection in Monkeys. II. Clinical Laboratory Responses to Heterologous Infection. Infect Dis. 128 (1) (1973), pp. 15-22. Jelinek, 2000. Jelinek, T. Dengue fever in international travellers. Clin Infect Dis 31(2000), pp. 144-147. Joshi et al, 2002. Joshi, V., Mourya, D.T., Sharma, R. C. Persistence of dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. Am J Trop Med Hyg 67 (2) (2002), pp.158-161. Khin and Thin 1983. M. M., Khin, M. M., Thin A. T. Transovarial transmission of dengue-2 virus by Aedes aegypti in nature. Am J Trop Med Hyg 32 (1983), pp.590594. Lanciotti et al, 1992. Lanciotti, R.S., Calisher, C. H., Gubler, D.J., Chang, G.J., Vorndam, V. Rapid detection and typing of dengue viruses from clinical samples by using Reverse Transcriptase-Polymerase Chain Reaction. J Clin Microbio 30 (1992), pp.545-551. Rosen and Gubler 1974. Rosen, I., Gubler, D. J. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23 (1973), pp. 1153-1157. Seah CLK et al, 1995. Seah, C.L.K., Chow V.T.K., Chan Y.C. Semi nested PCR using NS3 primers for the detection of dengue viruses in clinical serum specimens. Clin Diag virolo 4 (1995), pp.113-120. Shroyer 1990. Shroyer, D.A. Vertical maintenance of dengue-1 virus in sequential generations of Aedes albopictus. J Am Mosq Control Assoc 6 (1990), pp.312-314. World Health Organization.2009. Dengue: Guidelines for diagnosis, treatment, prevention and control: new edition. Geneva, Switzerland: WHO.
Wichmann 2005. Wichmann, O. Dengue antibody prevalence in German travellers. Emerg Infect Dis 11 (2005), pp.762-765.
Table. Details of multiple Dengue virus types (D-1, 2, 3 & 4) in individual mosquitoes of the 31 disease endemic settings of Rajasthan, India employing RTPCR assay Distri ct codes
1
Numbe r of individ ual infected mosqui toes process ed of each district 7
Dengue virus types (D-1, 2, 3 & 4) observed in individual mosquitoes (Mosq) as expressed in agarose gel Mosq 1
Mosq 2
Mosq 3
Mosq 4
Mosq 5
Mosq6
Mosq 7
Mosq 8
Mosq 9
Mosq 10
Total number of mosquitoes identified with multiple strains infectivity
D-2, 3 D-2, 3 D-1, 2, 3 D-3, 4 D-1, 2, 3, 4 D-3, 4 D-2, 3, 4 D-4
D-2, 3 D-3
D-2, 4 D-3
D-3, 4 D-1
D-2,3, 4
█
█
█
7
D-1
D-1, 2, 3 █
█
█
█
2
D-2, 3 D-1, 2, 3 D-3, 4 D-3, 4 D-3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 2 D-2, 4 D-2, 3 D-3, 4 D-1, 4 D-1, 2, 3, 4 D-4
D-3, 4 D-2, 3, 4 D-2, 3 D-2, 3 D-1, 2 D-3, 4 D-1, 3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 3, 4 D-1, 2, 3, 4 D-2, 3, 4 D2,3, 4 D-1, 2, 3, 4 D-1, 2, 3 D-2
2
6
3
7
D-2, 3 D-3, 4 D-2, 4 D-1, 2, 3, 4 D-1, 3, 4 D-4
D-1, 2, 3 D-2, 3, 4 D-3
D-2, 3
D-3
█
█
█
6
4
7
D-1, 2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3, 4 D-3, 4
D-1, 2, 3, 4 D-1, 2, 3 D-3, 4 D-3, 4 D-2, 3, 4 D-3, 4 █
█
█
█
7
5
7
█
█
█
6
6
10
D-1, 2 D-1, 2 █
D-1, 2, 4 D-1, 2, 4 █
9
7
10
8
7
9
7
█
█
█
6
10
6
█
█
█
6
11
7
D-2, 3,4 █
█
█
█
5
12
6
13
7
14
6
15
3
16
6
D-1, 2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3 D-2, 3, 4 D-2
█
█
█
6
D-1, 3, 4 █
█
█
█
7
█
█
█
6
█
█
█
█
3
█
█
█
█
2
17
7
D-2,3
D-2,3
D-2,3
D-3,4
D-2,
█
█
█
7
D-2, 3 D-2, 3, 4 D-2
D-2 D-2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3, 4 D-3, 4 D-3, 4 █ D-4
D-3 D-3 D-2, 3, 4 D-2, 3, 4 D-2, 4 D-4
D-2, 3,4
D-3, 4 D-2, 3, 4 D-2, 3, 4 █
D-2, 3, 4 D-2,3, 4
D-2, 3 D-2,
D-2, 3, 4 D-1,4
D-2, 3,4 D-1, 2, 3, 4 D-1,2,4
D-2, 3 █
D-2, 4 D-1, 2, 3,4 █
9 5
18
7
19
7
20
8
21
7
22
7
23
6
24
7
25
7
26
7
27
6
28
7
29
7
30
6
31
7
Total
D-1, 2, 3,4 D-1 D-2, 3,4 D-2, 3, 4 D-2, 3, 4 D-2 D-2, 3,4 D-2, 3 D-2 D-2, 3, 4 D-2, 3,4 D-2, 3, 4 D-1, 2, 3, 4 D-2, 3, 4
D-3, 4 D-2, 3 D-2, 3 D-2, 3, 4 D-2
D-1, 2, 4 D-2,3, 4 D-2, 4 D-1, 3, 4 D-2,3
D-2, 3 D-2
D-2
D-2 D-1, 2,3,4 D-2 D-1, 2, 3, 4 D-2 D-1, 2, 3, 4 D-2
D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 2, 3 D-1, 2, 3, 4
D-2, 4 D-1, 2, 3, 4 D-2, 3 D-2, 4 D-2, 3, 4 D-2, 3, 4 D-4
█
█
6
█
█
█
5
D-2, 3 █
█
█
8
█
█
7
█
█
█
5
█
█
█
3
D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 █
█
█
█
3
█
█
█
3
█
█
█
5
█
█
█
4
D-2
█
█
█
4
D-2
D-2
█
█
█
3
D-2, 3, 4 D-1,2,3, 4
█
█
█
█
6
D-1, 2, 3, 4
█
█
█
6
D-3, 4 D-2,3, 4
D-2 D-2
D-1, 2, 3, 4 D-4
D-4
D-4
D-2
D-2, 3, 4 D-2, 3, 4 D-2
D-2
D-2,3, 4
D-2
D-2, 3, 4 D-2
D-2, 3, 4 D-2, 3, 4 D-2, 3
█= No mosquito processed; D= Dengue strain
D-2, 3 D-2 D-2, 3 D-1, 2, 3
3 D-2, 3 D-1, 2, 3, 4 D-2, 3, 4 D-2, 3 D-3, 4 █
█
D-4
D-2, 3, 4 D-2, 3 D-2
212
Graphical abstract
3,4 D-2, 3 D-2
D-3, 4 D-2, 3
167 (78.7%)
S0001-706X(15)30056-5 http://dx.doi.org/doi:10.1016/j.actatropica.2015.07.007 ACTROP 3675
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
14-1-2015 2-7-2015 9-7-2015
Please cite this article as: Angel, Bennet, Angel, Annette, Joshi, Vinod, Multiple dengue virus types harboured by individual mosquitoes.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multiple dengue virus types harboured by individual mosquitoes
Bennet Angel, Annette Angel and Vinod Joshi* Laboratory of Virology & Molecular Biology, Desert Medicine Research Centre, Indian Council of Medical Research, Jodhpur, India *Corresponding author: Dr. Vinod Joshi, Laboratory of virology & Molecular Biology, Desert Medicine Research Centre, Pali Road, Jodhpur-342 005, India. Telephone: 0912912729729; 09660767977; Fax: 0912912720618 E-mail: [email protected] Highlights
Multiple DENV types are present in individual mosquitoes from Rajasthan, India
The mosquitoes could be non human source of hetero DENV types in endemic settings.
Paper sensitizes further studies on multiple DENV types in mosquitoes and DHF.
Abstract The existing knowledge on pathogenesis and aetiology of DHF establishes that Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) are caused by two subsequent infections of two different serotypes of dengue affecting a common human population with a time gap. Present studies have been undertaken on 212 laboratory reared infected individual mosquitoes from larvae collected from 31 dengue endemic towns of Rajasthan, India. Type specific DEN viruses were detected from individual mosquitoes employing RT-PCR. In 78.7% of 212 infected individual mosquitoes studied, vertically transmitted multiple DENV types were observed. We report for the first time that single mosquitoes contain multiple dengue virus types. Keywords: Multiple dengue types; Etiology; DHF
Dengue Fever (DF) with Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) is an emerging public health problem with an estimated number of 390 million cases reported annually (Bhatt and Brady, 2013). The severe form of dengue ie, DHF or DSS, responsible for case fatality up to more than 5% (Gubler, 2011) is believed to be associated with an Antibody Dependent Enhancement (ADE) in the patients (Halstead and Rourke, 1977), possibly caused by infectious immune complexes formed between a current DENV infection and non neutralizing antibodies already present in the patient’s system (Guzman et al., 2010). As of today we stand with an understanding that multiple infections of more than one DENV types with a time gap is a pre-requisite for DHF. To understand infection of different DENV types in a population with a gap of time, more emphasis has been given so far on isolation of DENV types (DEN-1, 2, 3 or 4) from the human patients (Guzman, 1981). Although dengue transmission involves both, horizontal as well as vertical transmission of virus (WHO, 2009), latter has not been given adequate importance. Mosquitoes, in addition to transmit virus from man to man, may also contribute extrinsic or unchallenged viral stock passaged by its own system by virtue of transovarial transmission of virus (Khin and Than, 1983; Rosen and Gubler, 1974; Joshi etal., 2002). We hypothesized that studies to comprehend the DENV types contained by single mosquito in a setting could add to our knowledge on non human source of multiple dengue types in an endemic area. During the course of present investigations we have undertaken molecular isolation of dengue virus types from individual specimens of laboratory reared Aedes aegypti from larvae collected from 31 disease endemic districts towns of Rajasthan state, India. The paper reports first observations on presence of multiple dengue virus types carried by individual
mosquitoes and discusses the possible significance of the observations in aetiology of DHF.
Studies were undertaken in all 33 district headquarter towns of north-western state Rajasthan, India. Rajasthan represents the arid ecosystem forming western boarder of India. It includes 33 districts which form a north-western desert part, an eastern semiarid part and southern hilly area. Out of 33 district headquarter towns, 28 are endemic for dengue fever (Unpublished data, state health department, Government of Rajasthan, India). The areas from where larval mosquitoes were collected are 31 district headquarter towns as listed at the foot note (Table). No breeding was seen in two districts i.e. Udaipur and Dholpur. Overall a total of 19,775 households were surveyed.
Larvae of Aedes aegypti were collected from the domestic and peri-domestic water collections from different localities in the urban settings of 31 dengue endemic districts of state of Rajasthan, India from May, 2012 till May 2014 using random sampling design. Random numbers were assigned to selected houses in study towns. Depending upon the size of town, every 6th, 8th or 10th house was chosen. All the breeding containers available in a sampled house were examined for larval collection. A total of 1, 30, 525 containers were surveyed. The larvae were brought and reared in laboratory into adult mosquitoes at ambient temperature of about 25˚C. Larval food of dog biscuit and yeast powder was provided. Adult Aedes aegypti emerged were kept in Barraud cages maintained individually for each locality in each district for about 56 days at about 25˚C and relative humidity of 65%.
Individual Aedes adults were homogenized in 200µl PBS and centrifuged at 5000 rpm for 10 minutes at 4˚C. The supernatant were transferred to clean eppendorfs, labelled district wise and stored at -80˚C. For screening the presence or absence of dengue virus antigen within them, 5µl of this supernatant (before storing) was subjected to Indirect Fluorescence Antibody test (IFAT) and a green fluorescence observed was recorded as IFAT positive result.
Thus a total number of 5136 laboratory reared mosquitoes were screened employing Indirect Fluorescence Antibody Test (IFAT), of which 1263 (24.59%) were found positive by dengue virus. Of these 1263 vertically infected mosquitoes, 212 specimens were selected (6-10 mosquitoes from each setting) and subjected to DENV type virus detection employing RT-PCR.
Viral RNA was extracted using the QIAmp Viral RNA kit (m/s Qiagen Inc., Valencia, CA). Gene specific primers (Lanciotti et al, 1992) were used for detecting the four types of DEN virus in the individual samples. These primers used were commercially synthesized from m/s Eurofins, Bangalore, India. Thus for each sample a total of 4 reaction was run, one representing a single type. The Superscript III RT/Taq Polymerase One-Step RT-PCR kit (manufactured by m/s Invitrogen, USA) was used for the assay following the manufacturer’s protocol. The temperature conditions were also as suggested in the protocol. After the amplification, 10µl of PCR product (four for each sample) was run on a 2% Agarose gel (m/s Invitrogen, USA) and then viewed in the Gel Documentation System (manufactured by m/s Bio-Rad, USA). This was done for a total of 212 individual mosquitoes. The types that appeared as bands were noted for each sample. Of these, eight mosquitoes were selected for sequencing.
Besides these 8 mosquitoes, viral suspensions of 6 infected mosquitoes were inoculated in Cell lines of C6/36 (Aedes albopictus) procured commercially from National Centre for Cell Sciences (NCCS), Pune, India. 420 µl of this suspension was subjected to RNA extraction and RT-PCR amplification as mentioned above. The primers used were as that referred in Lanciotti et al, 1992. These cell-line inoculated mosquito samples were also reacted with another set of type-specific primers (Seah et al, 1995). After detecting the type-specific bands for each sample in the Agarose gel, the remaining sample was purified using the PCR purification kit (manufactured by m/s Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The final purified product was eluted in 10 µl of Elution buffer provided in the kit. Thus a total of 14 samples of infected mosquitoes showing multiple DEN-V types were subjected to sequencing.1-2 µl of this PCR purified product was subjected to cycle sequencing using the Big Dye Terminator cycle sequencing kit v3.1 (manufactured by m/s Invitrogen, USA). This was followed by purification of the cycle sequenced product using the Dye-Ex 2.0 spin kit (manufactured by m/s Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The purified product was then loaded in the 16-capillary based 3130XL Genetic Analyzer (m/s ABI, USA).
Data generated from the sequencer were then analyzed using the ‘Sequence Analysis’ software. The quality check and trimming of the sequences generated was done using the ‘Seqscape 2.6’ software (m/s Applied Biosystems, USA) and then attempted for their assembly with the available dengue virus type-specific reference genomes. After trimming, BLAST was carried out. Further, multiple alignment was also performed using the ‘Clustal-W’ for the samples by placing them in four categories according to their types identified (Den-1, 2, 3 or 4).
Hence in all, a total number of 1, 30, 525 domestic water containers from 31 district towns of Rajasthan, India were searched for larval collection of Aedes mosquitoes. Total number of 5136 laboratory reared mosquitoes were screened for IFAT of which 1263 (24.59%) were found positive by vertically transmitted dengue virus. Of these, 212 (174 females and 38 males) specimens were selected from 31 study settings (610 mosquitoes from each district) for type-specific RT-PCR. Table shows results of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for DENV types as observed in individual mosquitoes. It was observed that in 78.7% of total specimens studied, more than one DEN type was present. Further analysis of data of mixed infections showed that in 163 out of 212 mosquitoes (76.8%) DEN-2 was observed, in 155 (73.1%) DEN-3 was present, in 132 (62.2%) DEN-4 was present and least 53 (25%) specimens DEN-1 was observed. The sequences of the 14 samples (processed for sequencing), obtained after trimming when assembled and annotated with the reference
genomes
of
the
four
genotypes
(NC_001477.1,
NC_001474.2,
NC_001475.2, NC_002640.1 respectively) gave no match. But when these sequences were matched and aligned with each other using Clustal-W software showed good coverage and identity. The results of this multiple sequence alignment showed presence of multiple DEN-V types in 6 of 14 mosquito samples while rest showed single or no DEN-V types. Of these, one mosquito sample showed good coverage for all the four DEN-V types, 5 showed good coverage for two DEN-V types and five showed coverage for single DEN-V type. The sequencing results of the six mosquitoes inoculated in cell lines and amplified with second set of primers (Seah et al, 1995) showed good assembly and annotation with DENV-2. The sequences generated will be attempted for submission in the NCBI website.
It is believed that primary infection by any of the serotypes of dengue leads to simple dengue fever (Guzman et al., 2010), whereas infection by two different dengue strains with a time interval may lead to DHF/DSS (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000). Experimental basis of above hypothesis is the DENV type specific virus isolations from human population cohort exhibiting DHF/DSS. In addition, the observations on experimental infection of monkeys by different DENV types with time intervals have also contributed to strengthen above hypothesis (Halstead et al., 1973). However, limitations of the above studies (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000) is that DENV types have been observed from serum samples of cohort of human patients which represents an immunologically challenged virus stock. Limitations of the other studies in supporting above hypothesis is that earlier workers (Halstead et al., 1973), have performed experiments on 118 rhesus monkeys but only one animal has manifested signs of DHF viz; thrombocytopenia and elevation of prothrombin, when challenged by different DENV types with a time interval. The aetiology and pathogenesis of DHF/DSS are still not fully understood (Gubler, 1998). Although earlier workers (Lanciotti et al., 1992; Wichmann, 2005; Jelinek, 2000) have reported enough serological evidences to explain pathogenesis of DHF, however none of the studies has included study of DENV types from mosquitoes. It is well known that dengue viruses undergo transovarial transmission (Khin and Khin, 1983; Rosen and Gubler, 1974). Experimental studies have also shown persistence of transovarial transmission in successive generations of infected mosquitoes (Joshi et al., 2002; Halstead et al., 1973; Shroyer, 1990).
We
hypothesized that transovarial transmission of dengue virus, in addition to serve as the possible mechanism of virus retention in nature (Joshi et al., 2002), may also conserve the hetero DENV types in study settings. Based on our observations of detection of
DENV types from 212 individual infected mosquitoes from 31 disease endemic settings of Rajasthan, India of which 78.7% carried more than one DENV type. We report for the first time that vertically infected individual mosquitoes possess multiple DNV types.
Present observations highlight two distinct messages. One is that in dengue endemic settings, through transovarial route, multiple DENV types establish themselves across mosquito generations. Since we have made RT-PCR virus isolations on individual mosquitoes and not on the pools of mosquitoes, these observations carry their own significance to think whether repetitive bites of mosquitoes infected by multiple DENV types could serve to provide source of hetero DENV types
in endemic
settings. Present observations get supported by the earlier reports citing that among young children even primary infection of dengue leads to vascular leakage and that age dependant host factors contribute to such manifestations (Alvarez, 2006; Guzman, 2002; Gamble 2000).
Acknowledgements We thank Director, Desert Medicine Research Centre (Indian Council of Medical Research), Jodhpur for providing facilities and support in conducting above work. Present work was supported by the Indian Council of Medical Research, Ministry of Health & Family Welfare, Government of India, India. We also thank Sh. Ajay Prakash Joshi, Scientist-I, Dr. Rajendra Kumar Baharia, Scientist-II and Smt. Suman Rathore, Research Assistant, Desert Medicine Research Centre, Jodhpur for providing support for Bioinformatics analysis.
References Alvarez, 2006. Alvarez M. Dengue hemorrhagic fever caused by sequential dengue 13 virus infections over a long time interval: Havana epidemic 2001-2002. Am J Trop Med Hyg 75 (2006), pp. 1113-1117. Bhatt et al, 2013. Bhatt, S. and P. W. Gething, O. J. Brady. The global distribution and burden of dengue. Nature 496 (2013), pp. 504-507. Gubler, 1998. Gubler, D. J. Dengue and Dengue Hemorrhagic Fever. Cli Micro Rev 11(1998), pp. 480-496. Gamble, 2000. Gamble, J. Age related changes in microvascular permeability: a significant factor in susceptibility of children to shock. 2000. Clin Sci 98 (2000), pp.211-216. Guzman, 2002. Guzman, M.G. Enhanced severity of secondary dengue-2 infections. Death rates in 1981 and 1997 Cuban outbreaks. Rev Panam Salud Publica 11 (2002), pp. 223-227. Guzman et al., 2010. Guzman, M. G., Halstead, S. B, Artsob, H., Buchy, P. Dengue: A continuing global threat. Nature: (2010), pp. 7-16. Gubler, 2011. Gubler, D. J. Dengue, Urbanization and globalization: The unholy treaty of 21st century. Trop Med Hlth 39 (2011), pp. 3-11. Guzman, 1990. Guzman, M. G. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seo epidemiologic study. 1990. Am J Trop Med Hyg 42 (1990), pp. 179184. Halstead and O’Rourke, 1977. Halstead, S. B., O’Rourke, E. J. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non neutralizing antibody. J Exp med 146 (1977), pp. 201-217. Halstead et al, 1973. Halstead, S.B., Henry, S., Jordi, C. Studies on the Pathogenesis of Dengue Infection in Monkeys. II. Clinical Laboratory Responses to Heterologous Infection. Infect Dis. 128 (1) (1973), pp. 15-22. Jelinek, 2000. Jelinek, T. Dengue fever in international travellers. Clin Infect Dis 31(2000), pp. 144-147. Joshi et al, 2002. Joshi, V., Mourya, D.T., Sharma, R. C. Persistence of dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. Am J Trop Med Hyg 67 (2) (2002), pp.158-161. Khin and Thin 1983. M. M., Khin, M. M., Thin A. T. Transovarial transmission of dengue-2 virus by Aedes aegypti in nature. Am J Trop Med Hyg 32 (1983), pp.590594. Lanciotti et al, 1992. Lanciotti, R.S., Calisher, C. H., Gubler, D.J., Chang, G.J., Vorndam, V. Rapid detection and typing of dengue viruses from clinical samples by using Reverse Transcriptase-Polymerase Chain Reaction. J Clin Microbio 30 (1992), pp.545-551. Rosen and Gubler 1974. Rosen, I., Gubler, D. J. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23 (1973), pp. 1153-1157. Seah CLK et al, 1995. Seah, C.L.K., Chow V.T.K., Chan Y.C. Semi nested PCR using NS3 primers for the detection of dengue viruses in clinical serum specimens. Clin Diag virolo 4 (1995), pp.113-120. Shroyer 1990. Shroyer, D.A. Vertical maintenance of dengue-1 virus in sequential generations of Aedes albopictus. J Am Mosq Control Assoc 6 (1990), pp.312-314. World Health Organization.2009. Dengue: Guidelines for diagnosis, treatment, prevention and control: new edition. Geneva, Switzerland: WHO.
Wichmann 2005. Wichmann, O. Dengue antibody prevalence in German travellers. Emerg Infect Dis 11 (2005), pp.762-765.
Table. Details of multiple Dengue virus types (D-1, 2, 3 & 4) in individual mosquitoes of the 31 disease endemic settings of Rajasthan, India employing RTPCR assay Distri ct codes
1
Numbe r of individ ual infected mosqui toes process ed of each district 7
Dengue virus types (D-1, 2, 3 & 4) observed in individual mosquitoes (Mosq) as expressed in agarose gel Mosq 1
Mosq 2
Mosq 3
Mosq 4
Mosq 5
Mosq6
Mosq 7
Mosq 8
Mosq 9
Mosq 10
Total number of mosquitoes identified with multiple strains infectivity
D-2, 3 D-2, 3 D-1, 2, 3 D-3, 4 D-1, 2, 3, 4 D-3, 4 D-2, 3, 4 D-4
D-2, 3 D-3
D-2, 4 D-3
D-3, 4 D-1
D-2,3, 4
█
█
█
7
D-1
D-1, 2, 3 █
█
█
█
2
D-2, 3 D-1, 2, 3 D-3, 4 D-3, 4 D-3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 2 D-2, 4 D-2, 3 D-3, 4 D-1, 4 D-1, 2, 3, 4 D-4
D-3, 4 D-2, 3, 4 D-2, 3 D-2, 3 D-1, 2 D-3, 4 D-1, 3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 3, 4 D-1, 2, 3, 4 D-2, 3, 4 D2,3, 4 D-1, 2, 3, 4 D-1, 2, 3 D-2
2
6
3
7
D-2, 3 D-3, 4 D-2, 4 D-1, 2, 3, 4 D-1, 3, 4 D-4
D-1, 2, 3 D-2, 3, 4 D-3
D-2, 3
D-3
█
█
█
6
4
7
D-1, 2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3, 4 D-3, 4
D-1, 2, 3, 4 D-1, 2, 3 D-3, 4 D-3, 4 D-2, 3, 4 D-3, 4 █
█
█
█
7
5
7
█
█
█
6
6
10
D-1, 2 D-1, 2 █
D-1, 2, 4 D-1, 2, 4 █
9
7
10
8
7
9
7
█
█
█
6
10
6
█
█
█
6
11
7
D-2, 3,4 █
█
█
█
5
12
6
13
7
14
6
15
3
16
6
D-1, 2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3 D-2, 3, 4 D-2
█
█
█
6
D-1, 3, 4 █
█
█
█
7
█
█
█
6
█
█
█
█
3
█
█
█
█
2
17
7
D-2,3
D-2,3
D-2,3
D-3,4
D-2,
█
█
█
7
D-2, 3 D-2, 3, 4 D-2
D-2 D-2, 3, 4 D-1, 2, 3, 4 D-1, 2, 3, 4 D-3, 4 D-3, 4 █ D-4
D-3 D-3 D-2, 3, 4 D-2, 3, 4 D-2, 4 D-4
D-2, 3,4
D-3, 4 D-2, 3, 4 D-2, 3, 4 █
D-2, 3, 4 D-2,3, 4
D-2, 3 D-2,
D-2, 3, 4 D-1,4
D-2, 3,4 D-1, 2, 3, 4 D-1,2,4
D-2, 3 █
D-2, 4 D-1, 2, 3,4 █
9 5
18
7
19
7
20
8
21
7
22
7
23
6
24
7
25
7
26
7
27
6
28
7
29
7
30
6
31
7
Total
D-1, 2, 3,4 D-1 D-2, 3,4 D-2, 3, 4 D-2, 3, 4 D-2 D-2, 3,4 D-2, 3 D-2 D-2, 3, 4 D-2, 3,4 D-2, 3, 4 D-1, 2, 3, 4 D-2, 3, 4
D-3, 4 D-2, 3 D-2, 3 D-2, 3, 4 D-2
D-1, 2, 4 D-2,3, 4 D-2, 4 D-1, 3, 4 D-2,3
D-2, 3 D-2
D-2
D-2 D-1, 2,3,4 D-2 D-1, 2, 3, 4 D-2 D-1, 2, 3, 4 D-2
D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 D-1, 2, 3 D-1, 2, 3, 4
D-2, 4 D-1, 2, 3, 4 D-2, 3 D-2, 4 D-2, 3, 4 D-2, 3, 4 D-4
█
█
6
█
█
█
5
D-2, 3 █
█
█
8
█
█
7
█
█
█
5
█
█
█
3
D-2, 3, 4 D-2, 3, 4 D-2, 3, 4 █
█
█
█
3
█
█
█
3
█
█
█
5
█
█
█
4
D-2
█
█
█
4
D-2
D-2
█
█
█
3
D-2, 3, 4 D-1,2,3, 4
█
█
█
█
6
D-1, 2, 3, 4
█
█
█
6
D-3, 4 D-2,3, 4
D-2 D-2
D-1, 2, 3, 4 D-4
D-4
D-4
D-2
D-2, 3, 4 D-2, 3, 4 D-2
D-2
D-2,3, 4
D-2
D-2, 3, 4 D-2
D-2, 3, 4 D-2, 3, 4 D-2, 3
█= No mosquito processed; D= Dengue strain
D-2, 3 D-2 D-2, 3 D-1, 2, 3
3 D-2, 3 D-1, 2, 3, 4 D-2, 3, 4 D-2, 3 D-3, 4 █
█
D-4
D-2, 3, 4 D-2, 3 D-2
212
Graphical abstract
3,4 D-2, 3 D-2
D-3, 4 D-2, 3
167 (78.7%)