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A portable approach for the surveillance of dengue virus-infected mosquitoes
Journal of Virological Methods 183 (2012) 90–93
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
A portable approach for the surveillance of dengue virus-infected mosquitoes David A. Muller a , Francesca D. Frentiu c , Alejandra Rojas a , Luciano A. Moreira d , Scott L. O’Neill b,c , Paul R. Young a,b,∗ a
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia d FIOCRUZ/Centro de Pesquisas René Rachou, Av. Augusto de Lima, 1715 Barro Preto, Belo Horizonte, Brazil b c
a b s t r a c t Article history: Received 11 January 2012 Received in revised form 15 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Dengue virus Mosquito Field surveillance Virus detection
Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses. © 2012 Elsevier B.V. All rights reserved.
Dengue virus (DENV) causes over 100 million human infections each year, with almost half of the world’s population at risk of contracting the virus. Infection with DENV can result in a range of outcomes from asymptomatic infection to clinical manifestations ranging from dengue fever through to the life threatening complications of dengue hemorrhagic fever and shock (Guzman et al., 2010; Malavige et al., 2004; Ross, 2010). The virus is transmitted to humans by the mosquito Aedes aegypti and, to a much lesser extent, by its congener Aedes albopictus through the bite of an infectious female. In the absence of effective alternatives, current disease control measures focus on the suppression of mosquito populations to reduce virus transmission. Preventing the onset of dengue outbreaks and effective vector management require surveillance of DENV prevalence in natural mosquito populations. Current methods of detecting DENV in field-caught mosquitoes either require virus isolation, followed by propagation in cell culture and detection using ELISA, or the extraction of viral RNA and RT-PCR (Ahmad et al., 1997; Chan et al., 1994; Chow et al., 1998; Hall-Mendelin
∗ Corresponding author at: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. Tel.: +61 7 33654646; fax: +61 7 33654620. E-mail address: [email protected] (P.R. Young). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.03.033
et al., 2010; Kuberski and Rosen, 1977; Samuel and Tyagi, 2006; Sithiprasasna et al., 1994). These methods are time consuming and require specialized equipment and highly trained personnel that are not always available in developing regions of the world where dengue imposes a high health and economic burden. Where they are available, they are located in centralized facilities requiring coordinated collection and transport of trapped mosquitoes and processing before release of data for incorporation into surveillance reports. DENV, a member of the Flaviviridae family and genus Flavivirus, is a small enveloped virus with an 11 kb single-stranded positivesense RNA genome encoding 3 structural and 7 non-structural proteins (Lindenbach and Rice, 2003). NS1 is a 352-amino acid non-structural protein that is 46-55 kDa in size depending on glycosylation status (Deubel et al., 1988; Mackow et al., 1987; Wright et al., 1989). It exists in multiple oligomeric forms and is found at different cellular locations, either membrane-associated in vesicular compartments within the cell or on the cell surface and as a secreted extracellular (nonvirion) form (sNS1) (Mason, 1989; Smith and Wright, 1985; Westaway and Goodman, 1987; Winkler et al., 1988). The secreted form of NS1 has become a widely used serological biomarker for DENV infection because it is found in patient sera from the onset of illness though to 12 days post-onset, in quantities of up to 50 g/ml (Alcon et al., 2002; Castro-Jorge et al., 2010;
D.A. Muller et al. / Journal of Virological Methods 183 (2012) 90–93
Lima et al., 2010; Muller and Young, 2011; Wang and Sekaran, 2010a; Young et al., 2000). Several NS1-based diagnostic assays are currently available commercially, in both rapid strip lateral flow and ELISA formats, for the detection of DENV in human sera (Blacksell et al., 2008; Kumarasamy et al., 2007a,b; Shu et al., 2009; Wang and Sekaran, 2010b; Zainah et al., 2009). The potential of a rapid NS1-based diagnostic assay as a portable and inexpensive tool for monitoring the prevalence of DENV in mosquitoes in the field was investigated. Genetically diverse female A. aegypti mosquitoes (generation 3 post-field collection from Cairns, Australia) were experimentally infected with DENV serotype 2 (strain ET-300, a low passage isolate from a patient infected in East Timor in 2000). Five to seven day old mosquitoes were injected in the thorax with 69 nl of virus at a titer of 1 × 106 plaque forming units/ml. Injection in the thorax circumvents the mosquito midgut barrier and ensures infection with the virus thereby minimizing the risk of false negatives. Following injection, mosquitoes were kept in cups under a 12L:12D light cycle, at 26 ◦ C and high relative humidity, and allowed to feed ad libitum on 10% sucrose. DENV2 ET-300 had been passaged four times in C6/36 cells prior to injections and titrated according to published protocols (Frentiu et al., 2010). Control, uninfected mosquitoes were maintained in parallel using the same conditions. Mosquitoes were collected at days 5, 10 and 15 post-infection by snap freezing and stored at −80 ◦ C. Mosquitoes were homogenized using one of two methods. In the first method, mosquitoes were placed in 5 ml tubes containing 2 ml of PBS and 4 mm diameter glass beads and homogenized for 2 min in a laboratory scale milling homogenizer (Mixer/Mill 8000, SPEX) (Fig. 1A). In the second method, individual mosquitoes were placed into a 1.5 ml microfuge tube containing 200 l of PBS and homogenized with a battery-operated hand-held motor with a disposable pestle (Pellet pestle motor, Kontes) (Fig. 1B). Homogenates used for NS1 detection were not clarified by centrifugation in order to simulate field conditions. NS1 quantitation was determined using the Panbio dengue early ELISA kit (catalog # E-DEN02P) with the addition of a standard curve (Young et al., 2000). Briefly, NS1 standards (recombinant baculovirus-derived NS1) and mosquito homogenates were diluted separately in antigen diluent and 100 l of sample was incubated for 1 h at 37 ◦ C. Following incubation, ELISA wells were washed six times in PBS (phosphate buffered saline) containing 0.05% Tween-20 (PBST-20) before the addition of a monoclonal antiNS1 IgG-HRP conjugate followed by incubation for 1 h at 37 ◦ C. Wells were washed a further six times before being developed for 10 min at room temperature with 100 l of tetramethylbenzidine (TMB). The reaction was stopped using 100 l of 1 M phosphoric acid and absorbance was read at 450 nm using a microplate spectrophotometer (SpectraMax, Molecular Devices). The time course of appearance and detection of NS1 in infected mosquito homogenates was investigated at 0, 5, 10 and 15 days post-infection (Fig. 1C). Similar levels were found in the homogenates at each of the 3 post-infection time points. The use of virus stocks prepared by harvesting media from infected C6/36 insect cell cultures ensured that any NS1 detected was not from the mosquito inoculum as NS1 is not secreted from infected insect cells (Mason, 1989; Muller and Young, 2011). Five days post-infection was chosen for subsequent experiments. Next, the amount of NS1 detected was compared using either of the two mosquito homogenization methods with experimentally infected mosquitoes. The amount of NS1 present in each mosquito was determined using the quantitative NS1 capture ELISA as described above. Both homogenization methods were effective at releasing NS1 from mosquitoes (5 mosquitoes per method) with an average of 88 ng/ml (milling machine) and 100 ng/ml (pestle) released (Fig. 1D). As both methods resulted in similar levels of NS1 release, the use of the NS1
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rapid strips for NS1 detection from mosquito homogenates was investigated. Following quantitation of NS1 levels by capture ELISA, rapid NS1 detection was performed using the Panbio dengue early rapid kit (Alere, catalog # R-DEN01P). Fifty l of mosquito homogenate was added to 25 l of reaction buffer and 25 l of the kit supplied anti-NS1 gold conjugated probe in a detection tube containing the rapid strip. The reaction was allowed to proceed for 15 min before the detection strips were visualized. Any sign of red in the detection area was indicative of a positive sample. As it would be impractical, time consuming and costly to test single trapped mosquitoes in the field, NS1 detection in pools of mosquitoes was also examined. To determine the limit of sensitivity of the assay for detection of NS1 in mosquito homogenates, one infected mosquito was homogenized in the following ratios of DENVinfected to uninfected mosquitoes: 1:10, 1:20, 1:30, 1:50 and 1:100 (Fig. 2). Tests were performed in triplicate. Using the portable pestle method for homogenization, NS1 was successfully detected in all pools containing an infected mosquito, including the 1:100 sample. However in the latter case the homogenate was too viscous to pipette into the reaction tube and so needed to be further diluted with PBS before detection. Given that minimal handling is desirable for practical purposes under field conditions, pools of mosquitoes no greater than 50 individuals should be examined. This method represents a simple and effective way of detecting NS1 from DENV-infected mosquitoes that could easily be applied to the surveillance of virus-infected mosquitoes trapped in the field. NS1 released in homogenates of infected mosquitoes prepared using a portable hand pestle was readily detected with a diagnostic NS1 rapid strip. NS1 detection in field-caught A. aegypti has recently been reported, providing proof-of-concept for field studies, but the method described in that report required specialized laboratory equipment and reagents unsuitable for onsite processing and detection (Tan et al., 2011). Using the approach outlined here, NS1 can be detected in pools of trapped mosquitoes with a crude extraction method that is rapid, requires no down-stream laboratory infrastructure or specialized staff, allows results to be obtained on-site in the field and for large numbers of mosquitoes to be processed in a single reaction. Currently, there are no other comparable approaches that would provide such rapid and specific pathogen detection in trapped mosquitoes in a field setting and consequently, as a first-in-class protocol it is difficult to benchmark its performance. Nevertheless, in comparison with the only currently available alternatives; laboratory based virus isolation, immunofluorescence in mosquito head-squashes and RT-PCR of pathogen specific nucleic acid from either mosquito extracts or honey-soaked cards on which mosquitoes have fed, the method described here is both more rapid and cost-effective. Although NS1 was readily detected at all sampling time points examined, the virus strain and replication kinetics in the mosquito may affect the sensitivity of the assay. In addition, there are a number of alternative NS1 strip formats available and these would need to be compared for specificity and sensitivity in the background of a mosquito homogenate rather than patient sera/plasma for which they were designed (Blacksell et al., 2011). Finally, the value of this approach for routine virus surveillance in natural mosquito populations can only be fully assessed in field evaluations during outbreaks of all four DENV serotypes as well as multiple circulating strains and these studies are currently being planned. Field assessment of trapped mosquitoes with this approach would allow both mosquito numbers and dengue infection status to be directly uploaded by field operators along with GIS information via PDAs or cell phones to a coordinating center for data processing. Both mosquito and dengue prevalence data could then be published
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D.A. Muller et al. / Journal of Virological Methods 183 (2012) 90–93
Fig. 1. (A) Laboratory based milling machine; (B) portable, hand held battery operated pestle with disposable end; (C) quantity of NS1 detected by NS1 capture ELISA in individual infected mosquitoes (n = 5) taken at 5, 10 and 15 days post infection and homogenized using the milling machine; (D) comparison of yields of NS1 released from individual dengue infected mosquitoes (n = 5) at 5 days post infection and homogenized by either the milling machine or the pestle motor.
Fig. 2. NS1 detection in dengue virus-infected mosquito pools. (A) Detection of NS1 using the Dengue Rapid test following homogenization of pools of infected and uninfected mosquitoes using the portable motorized pestle (*infected:uninfected mosquito ratios); (B) examples of rapid strips used for NS1 detection in mosquito homogenates. Left to right: uninfected mosquito control, single infected mosquito and 1 infected mosquito homogenized in a pool of 30 uninfected mosquitoes.
directly online for access by regional and local health officers. The availability of real-time monitoring of dengue presence within the mosquito population would be a valuable addition to early warning monitoring programs, resulting in more effective vector control responses.
Acknowledgments We thank Alere for the supply of the Panbio dengue Early Rapid and dengue Early ELISA kits and Dr. Alyssa Pyke for the DENV2 ET300 virus strain. This work was supported in part by the Australian Research Council as well as a grant from the National Institutes of
Health through the Grand Challenges in Global Health Initiative of the Bill and Melinda Gates Foundation. References Ahmad, R., Ismail, A., Saat, Z., Lim, L.H., 1997. Detection of dengue virus from field Aedes aegypti and Aedes albopictus adults and larvae. The Southeast Asian Journal of Tropical Medicine and Public Health 28, 138–142. Alcon, S., Talarmin, A., Debruyne, M., Falconar, A., Deubel, V., Flamand, M., 2002. Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. Journal of Clinical Microbiology 40, 376–381. Blacksell, S.D., Mammen Jr., M.P., Thongpaseuth, S., Gibbons, R.V., Jarman, R.G., Jenjaroen, K., Nisalak, A., Phetsouvanh, R., Newton, P.N., Day, N.P., 2008. Evaluation of the Panbio dengue virus nonstructural 1 antigen detection and
D.A. Muller et al. / Journal of Virological Methods 183 (2012) 90–93 immunoglobulin M antibody enzyme-linked immunosorbent assays for the diagnosis of acute dengue infections in Laos. Diagnostic Microbiology and Infectious Disease 60, 43–49. Blacksell, S.D., Jarman, R.G., Bailey, M.S., Tanganuchitcharnchai, A., Jenjaroen, K., Gibbons, R.V., Paris, D.H., Premaratna, R., de Silva, H.J., Lalloo, D.G., Day, N.P., 2011. Evaluation of six commercial point-of-care tests for diagnosis of acute dengue infections: the need for combining NS1 antigen and IgM/IgG antibody detection to achieve acceptable levels of accuracy. Clinical and Vaccine Immunology: CVI 18, 2095–2101. Castro-Jorge, L.A., Machado, P.R., Favero, C.A., Borges, M.C., Passos, L.M., de Oliveira, R.M., Fonseca, B.A., 2010. Clinical evaluation of the NS1 antigen-capture ELISA for early diagnosis of dengue virus infection in Brazil. Journal of Medical Virology 82, 1400–1405. Chan, S.Y., Kautner, I., Lam, S.K., 1994. Detection and serotyping of dengue viruses by PCR: a simple, rapid method for the isolation of viral RNA from infected mosquito larvae. The Southeast Asian Journal of Tropical Medicine and Public Health 25, 258–261. Chow, V.T., Chan, Y.C., Yong, R., Lee, K.M., Lim, L.K., Chung, Y.K., Lam-Phua, S.G., Tan, B.T., 1998. Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing. The American Journal of Tropical Medicine and Hygiene 58, 578–586. Deubel, V., Kinney, R.M., Trent, D.W., 1988. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome. Virology 165, 234–244. Frentiu, F.D., Robinson, J., Young, P.R., McGraw, E.A., O’Neill, S.L., 2010. Wolbachiamediated resistance to dengue virus infection and death at the cellular level. PLoS One 5, e13398. Guzman, M.G., Halstead, S.B., Artsob, H., Buchy, P., Farrar, J., Gubler, D.J., Hunsperger, E., Kroeger, A., Margolis, H.S., Martinez, E., Nathan, M.B., Pelegrino, J.L., Simmons, C., Yoksan, S., Peeling, R.W., 2010. Dengue: a continuing global threat. Nature Reviews Microbiology 8, S7–S16. Hall-Mendelin, S., Ritchie, S.A., Johansen, C.A., Zborowski, P., Cortis, G., Dandridge, S., Hall, R.A., van den Hurk, A.F., 2010. Exploiting mosquito sugar feeding to detect mosquito-borne pathogens. Proceedings of the National Academy of Sciences of the United States of America 107, 11255–11259. Kuberski, T.T., Rosen, L., 1977. A simple technique for the detection of dengue antigen in mosquitoes by immunofluorescence. The American Journal of Tropical Medicine and Hygiene 26, 533–537. Kumarasamy, V., Chua, S.K., Hassan, Z., Wahab, A.H., Chem, Y.K., Mohamad, M., Chua, K.B., 2007a. Evaluating the sensitivity of a commercial dengue NS1 antigencapture ELISA for early diagnosis of acute dengue virus infection. Singapore Medical Journal 48, 669–673. Kumarasamy, V., Wahab, A.H., Chua, S.K., Hassan, Z., Chem, Y.K., Mohamad, M., Chua, K.B., 2007b. Evaluation of a commercial dengue NS1 antigen-capture ELISA for laboratory diagnosis of acute dengue virus infection. Journal of Virological Methods 140, 75–79. Lima, Md.R.O., Nogueira, R.M.R., Schatzmayr, H.G., dos Santos, F.B.D., 2010. Comparison of three commercially available dengue NS1 antigen capture assays for acute diagnosis of dengue in Brazil. PLoS Neglected Tropical Diseases 4, e738. Lindenbach, B.D., Rice, C.M., 2003. Molecular biology of flaviviruses. Advances in Virus Research 59, 23–61.
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Mackow, E., Makino, Y., Zhao, B.T., Zhang, Y.M., Markoff, L., Buckler-White, A., Guiler, M., Chanock, R., Lai, C.J., 1987. The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins. Virology 159, 217–228. Malavige, G.N., Fernando, S., Fernando, D.J., Seneviratne, S.L., 2004. Dengue viral infections. Postgraduate Medical Journal 80, 588–601. Mason, P.W., 1989. Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169, 354–364. Muller, D.A., Young, P.R., 2011. The many faces of the flavivirus non-structural glycoprotein NS1. In: Shi, P.-Y. (Ed.), Molecular Virology and Control of Flaviviruses. Caister Academic Press, Norfolk, pp. 51–75. Ross, T.M., 2010. Dengue virus. Clinics in Laboratory Medicine 30, 149–160. Samuel, P.P., Tyagi, B.K., 2006. Diagnostic methods for detection & isolation of dengue viruses from vector mosquitoes. Indian Journal of Medical Research 123, 615–628. Shu, P.Y., Yang, C.F., Kao, J.F., Su, C.L., Chang, S.F., Lin, C.C., Yang, W.C., Shih, H., Yang, S.Y., Wu, P.F., Wu, H.S., Huang, J.H., 2009. Application of the dengue virus NS1 antigen rapid test for on-site detection of imported dengue cases at airports. Clinical Vaccine Immunology 16, 589–591. Sithiprasasna, R., Strickman, D., Innis, B.L., Linthicum, K.J., 1994. ELISA for detecting dengue and Japanese encephalitis viral antigen in mosquitoes. Annals of Tropical Medicine and Parasitology 88, 397–404. Smith, G.W., Wright, P.J., 1985. Synthesis of proteins and glycoproteins in dengue type 2 virus-infected vero and Aedes albopictus cells. Journal of General Virology 66 (Pt 3), 559–571. Tan, C.H., Wong, P.S., Li, M.Z., Vythilingam, I., Ng, L.C., 2011. Evaluation of the Dengue NS1 Ag Strip(R) for detection of dengue virus antigen in Aedes aegypti (Diptera: Culicidae). Vector-Borne and Zoonotic Diseases 11, 789–792. Wang, S.M., Sekaran, S.D., 2010a. Early diagnosis of Dengue infection using a commercial Dengue Duo rapid test kit for the detection of NS1, IGM, and IGG. American Journal of Tropical Medicine and Hygiene 83, 690–695. Wang, S.M., Sekaran, S.D., 2010b. Evaluation of a commercial SD dengue virus NS1 antigen capture enzyme-linked immunosorbent assay kit for early diagnosis of dengue virus infection. Journal of Clinical Microbiology 48, 2793–2797. Westaway, E.G., Goodman, M.R., 1987. Variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected Vero cells. Archives of Virology 94, 215–228. Winkler, G., Randolph, V.B., Cleaves, G.R., Ryan, T.E., Stollar, V., 1988. Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162, 187–196. Wright, P.J., Cauchi, M.R., Ng, M.L., 1989. Definition of the carboxy termini of the three glycoproteins specified by dengue virus type 2. Virology 171, 61–67. Young, P.R., Hilditch, P.A., Bletchly, C., Halloran, W., 2000. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. Journal of Clinical Microbiology 38, 1053–1057. Zainah, S., Wahab, A.H., Mariam, M., Fauziah, M.K., Khairul, A.H., Roslina, I., Sairulakhma, A., Kadimon, S.S., Jais, M.S., Chua, K.B., 2009. Performance of a commercial rapid dengue NS1 antigen immunochromatography test with reference to dengue NS1 antigen-capture ELISA. Journal of Virological Methods 155, 157–160.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
A portable approach for the surveillance of dengue virus-infected mosquitoes David A. Muller a , Francesca D. Frentiu c , Alejandra Rojas a , Luciano A. Moreira d , Scott L. O’Neill b,c , Paul R. Young a,b,∗ a
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia d FIOCRUZ/Centro de Pesquisas René Rachou, Av. Augusto de Lima, 1715 Barro Preto, Belo Horizonte, Brazil b c
a b s t r a c t Article history: Received 11 January 2012 Received in revised form 15 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Dengue virus Mosquito Field surveillance Virus detection
Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses. © 2012 Elsevier B.V. All rights reserved.
Dengue virus (DENV) causes over 100 million human infections each year, with almost half of the world’s population at risk of contracting the virus. Infection with DENV can result in a range of outcomes from asymptomatic infection to clinical manifestations ranging from dengue fever through to the life threatening complications of dengue hemorrhagic fever and shock (Guzman et al., 2010; Malavige et al., 2004; Ross, 2010). The virus is transmitted to humans by the mosquito Aedes aegypti and, to a much lesser extent, by its congener Aedes albopictus through the bite of an infectious female. In the absence of effective alternatives, current disease control measures focus on the suppression of mosquito populations to reduce virus transmission. Preventing the onset of dengue outbreaks and effective vector management require surveillance of DENV prevalence in natural mosquito populations. Current methods of detecting DENV in field-caught mosquitoes either require virus isolation, followed by propagation in cell culture and detection using ELISA, or the extraction of viral RNA and RT-PCR (Ahmad et al., 1997; Chan et al., 1994; Chow et al., 1998; Hall-Mendelin
∗ Corresponding author at: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. Tel.: +61 7 33654646; fax: +61 7 33654620. E-mail address: [email protected] (P.R. Young). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.03.033
et al., 2010; Kuberski and Rosen, 1977; Samuel and Tyagi, 2006; Sithiprasasna et al., 1994). These methods are time consuming and require specialized equipment and highly trained personnel that are not always available in developing regions of the world where dengue imposes a high health and economic burden. Where they are available, they are located in centralized facilities requiring coordinated collection and transport of trapped mosquitoes and processing before release of data for incorporation into surveillance reports. DENV, a member of the Flaviviridae family and genus Flavivirus, is a small enveloped virus with an 11 kb single-stranded positivesense RNA genome encoding 3 structural and 7 non-structural proteins (Lindenbach and Rice, 2003). NS1 is a 352-amino acid non-structural protein that is 46-55 kDa in size depending on glycosylation status (Deubel et al., 1988; Mackow et al., 1987; Wright et al., 1989). It exists in multiple oligomeric forms and is found at different cellular locations, either membrane-associated in vesicular compartments within the cell or on the cell surface and as a secreted extracellular (nonvirion) form (sNS1) (Mason, 1989; Smith and Wright, 1985; Westaway and Goodman, 1987; Winkler et al., 1988). The secreted form of NS1 has become a widely used serological biomarker for DENV infection because it is found in patient sera from the onset of illness though to 12 days post-onset, in quantities of up to 50 g/ml (Alcon et al., 2002; Castro-Jorge et al., 2010;
D.A. Muller et al. / Journal of Virological Methods 183 (2012) 90–93
Lima et al., 2010; Muller and Young, 2011; Wang and Sekaran, 2010a; Young et al., 2000). Several NS1-based diagnostic assays are currently available commercially, in both rapid strip lateral flow and ELISA formats, for the detection of DENV in human sera (Blacksell et al., 2008; Kumarasamy et al., 2007a,b; Shu et al., 2009; Wang and Sekaran, 2010b; Zainah et al., 2009). The potential of a rapid NS1-based diagnostic assay as a portable and inexpensive tool for monitoring the prevalence of DENV in mosquitoes in the field was investigated. Genetically diverse female A. aegypti mosquitoes (generation 3 post-field collection from Cairns, Australia) were experimentally infected with DENV serotype 2 (strain ET-300, a low passage isolate from a patient infected in East Timor in 2000). Five to seven day old mosquitoes were injected in the thorax with 69 nl of virus at a titer of 1 × 106 plaque forming units/ml. Injection in the thorax circumvents the mosquito midgut barrier and ensures infection with the virus thereby minimizing the risk of false negatives. Following injection, mosquitoes were kept in cups under a 12L:12D light cycle, at 26 ◦ C and high relative humidity, and allowed to feed ad libitum on 10% sucrose. DENV2 ET-300 had been passaged four times in C6/36 cells prior to injections and titrated according to published protocols (Frentiu et al., 2010). Control, uninfected mosquitoes were maintained in parallel using the same conditions. Mosquitoes were collected at days 5, 10 and 15 post-infection by snap freezing and stored at −80 ◦ C. Mosquitoes were homogenized using one of two methods. In the first method, mosquitoes were placed in 5 ml tubes containing 2 ml of PBS and 4 mm diameter glass beads and homogenized for 2 min in a laboratory scale milling homogenizer (Mixer/Mill 8000, SPEX) (Fig. 1A). In the second method, individual mosquitoes were placed into a 1.5 ml microfuge tube containing 200 l of PBS and homogenized with a battery-operated hand-held motor with a disposable pestle (Pellet pestle motor, Kontes) (Fig. 1B). Homogenates used for NS1 detection were not clarified by centrifugation in order to simulate field conditions. NS1 quantitation was determined using the Panbio dengue early ELISA kit (catalog # E-DEN02P) with the addition of a standard curve (Young et al., 2000). Briefly, NS1 standards (recombinant baculovirus-derived NS1) and mosquito homogenates were diluted separately in antigen diluent and 100 l of sample was incubated for 1 h at 37 ◦ C. Following incubation, ELISA wells were washed six times in PBS (phosphate buffered saline) containing 0.05% Tween-20 (PBST-20) before the addition of a monoclonal antiNS1 IgG-HRP conjugate followed by incubation for 1 h at 37 ◦ C. Wells were washed a further six times before being developed for 10 min at room temperature with 100 l of tetramethylbenzidine (TMB). The reaction was stopped using 100 l of 1 M phosphoric acid and absorbance was read at 450 nm using a microplate spectrophotometer (SpectraMax, Molecular Devices). The time course of appearance and detection of NS1 in infected mosquito homogenates was investigated at 0, 5, 10 and 15 days post-infection (Fig. 1C). Similar levels were found in the homogenates at each of the 3 post-infection time points. The use of virus stocks prepared by harvesting media from infected C6/36 insect cell cultures ensured that any NS1 detected was not from the mosquito inoculum as NS1 is not secreted from infected insect cells (Mason, 1989; Muller and Young, 2011). Five days post-infection was chosen for subsequent experiments. Next, the amount of NS1 detected was compared using either of the two mosquito homogenization methods with experimentally infected mosquitoes. The amount of NS1 present in each mosquito was determined using the quantitative NS1 capture ELISA as described above. Both homogenization methods were effective at releasing NS1 from mosquitoes (5 mosquitoes per method) with an average of 88 ng/ml (milling machine) and 100 ng/ml (pestle) released (Fig. 1D). As both methods resulted in similar levels of NS1 release, the use of the NS1
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rapid strips for NS1 detection from mosquito homogenates was investigated. Following quantitation of NS1 levels by capture ELISA, rapid NS1 detection was performed using the Panbio dengue early rapid kit (Alere, catalog # R-DEN01P). Fifty l of mosquito homogenate was added to 25 l of reaction buffer and 25 l of the kit supplied anti-NS1 gold conjugated probe in a detection tube containing the rapid strip. The reaction was allowed to proceed for 15 min before the detection strips were visualized. Any sign of red in the detection area was indicative of a positive sample. As it would be impractical, time consuming and costly to test single trapped mosquitoes in the field, NS1 detection in pools of mosquitoes was also examined. To determine the limit of sensitivity of the assay for detection of NS1 in mosquito homogenates, one infected mosquito was homogenized in the following ratios of DENVinfected to uninfected mosquitoes: 1:10, 1:20, 1:30, 1:50 and 1:100 (Fig. 2). Tests were performed in triplicate. Using the portable pestle method for homogenization, NS1 was successfully detected in all pools containing an infected mosquito, including the 1:100 sample. However in the latter case the homogenate was too viscous to pipette into the reaction tube and so needed to be further diluted with PBS before detection. Given that minimal handling is desirable for practical purposes under field conditions, pools of mosquitoes no greater than 50 individuals should be examined. This method represents a simple and effective way of detecting NS1 from DENV-infected mosquitoes that could easily be applied to the surveillance of virus-infected mosquitoes trapped in the field. NS1 released in homogenates of infected mosquitoes prepared using a portable hand pestle was readily detected with a diagnostic NS1 rapid strip. NS1 detection in field-caught A. aegypti has recently been reported, providing proof-of-concept for field studies, but the method described in that report required specialized laboratory equipment and reagents unsuitable for onsite processing and detection (Tan et al., 2011). Using the approach outlined here, NS1 can be detected in pools of trapped mosquitoes with a crude extraction method that is rapid, requires no down-stream laboratory infrastructure or specialized staff, allows results to be obtained on-site in the field and for large numbers of mosquitoes to be processed in a single reaction. Currently, there are no other comparable approaches that would provide such rapid and specific pathogen detection in trapped mosquitoes in a field setting and consequently, as a first-in-class protocol it is difficult to benchmark its performance. Nevertheless, in comparison with the only currently available alternatives; laboratory based virus isolation, immunofluorescence in mosquito head-squashes and RT-PCR of pathogen specific nucleic acid from either mosquito extracts or honey-soaked cards on which mosquitoes have fed, the method described here is both more rapid and cost-effective. Although NS1 was readily detected at all sampling time points examined, the virus strain and replication kinetics in the mosquito may affect the sensitivity of the assay. In addition, there are a number of alternative NS1 strip formats available and these would need to be compared for specificity and sensitivity in the background of a mosquito homogenate rather than patient sera/plasma for which they were designed (Blacksell et al., 2011). Finally, the value of this approach for routine virus surveillance in natural mosquito populations can only be fully assessed in field evaluations during outbreaks of all four DENV serotypes as well as multiple circulating strains and these studies are currently being planned. Field assessment of trapped mosquitoes with this approach would allow both mosquito numbers and dengue infection status to be directly uploaded by field operators along with GIS information via PDAs or cell phones to a coordinating center for data processing. Both mosquito and dengue prevalence data could then be published
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Fig. 1. (A) Laboratory based milling machine; (B) portable, hand held battery operated pestle with disposable end; (C) quantity of NS1 detected by NS1 capture ELISA in individual infected mosquitoes (n = 5) taken at 5, 10 and 15 days post infection and homogenized using the milling machine; (D) comparison of yields of NS1 released from individual dengue infected mosquitoes (n = 5) at 5 days post infection and homogenized by either the milling machine or the pestle motor.
Fig. 2. NS1 detection in dengue virus-infected mosquito pools. (A) Detection of NS1 using the Dengue Rapid test following homogenization of pools of infected and uninfected mosquitoes using the portable motorized pestle (*infected:uninfected mosquito ratios); (B) examples of rapid strips used for NS1 detection in mosquito homogenates. Left to right: uninfected mosquito control, single infected mosquito and 1 infected mosquito homogenized in a pool of 30 uninfected mosquitoes.
directly online for access by regional and local health officers. The availability of real-time monitoring of dengue presence within the mosquito population would be a valuable addition to early warning monitoring programs, resulting in more effective vector control responses.
Acknowledgments We thank Alere for the supply of the Panbio dengue Early Rapid and dengue Early ELISA kits and Dr. Alyssa Pyke for the DENV2 ET300 virus strain. This work was supported in part by the Australian Research Council as well as a grant from the National Institutes of
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