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Journal of the Formosan Medical Association (2015) xx, 1e7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan Tien-Huang Chen, Shu-Wan Jian, Chih-Yuan Wang, Cheo Lin, Pei-Feng Wang, Chien-Ling Su, Hwa-Jen Teng*, Pei-Yun Shu, Ho-Sheng Wu Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Number 161, Kunyang Street, Taipei 11561, Taiwan, ROC Received 17 August 2014; received in revised form 3 December 2014; accepted 26 December 2014
KEYWORDS Aedes aegypti; Aedes albopictus; Chikungunya virus; mosquito infection; Taiwan
Background/purpose: An E1/226V variant Chikungunya virus (CHIKV) efficiently transmitted by Aedes albopictus to humans poses a significant threat to public health for those areas with the presence of Aedes albopictus, including Taiwan. Methods: We infected three imported CHIKV isolates including the E1/226V variant with Ae. albopictus and Aedes aegypti in the laboratory to understand the disease risk. Viral RNA was measured by real time reverse transcription polymerase chain reaction. Results: The viral susceptibility varied by virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5e6 days post infection (dpi) with the maximum virus yield, ranging from 103.63 to 103.87 at 5e10 dpi in both species. The variant CHIKV Central/ East/South African (CESA) virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 105.63 to 106.52 at 3e6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1e2 dpi with the maximum virus yield ranging from 105.51 to106.27 at 6e12 dpi. In Ae. aegypti, these viruses replicated at 1e2 dpi, with maximum yields at 4 e5 dpi (range from 105.38 to 105.62). Conclusion: We concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype and that the E1/226V variant virus strain presents an even higher risk. Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Conflicts of interest: The authors have no conflicts of interest relevant to this article. * Corresponding author. Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Number 161, Kunyang Street, Taipei 11561, Taiwan, ROC. E-mail address: [email protected] (H.-J. Teng). http://dx.doi.org/10.1016/j.jfma.2014.12.002 0929-6646/Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Introduction Chikungunya virus (CHIKV) is a member of Alphavirus (Togaviridae) and is transmitted to human beings by Aedes mosquitoes. It was first isolated from a patient in Tanzania in 1953.1 Later, it caused numerous epidemic cases in Africa and Asia.2,3 This virus has urban and sylvatic transmission cycles, which have their own vector species. The main vector species responsible for urban transmission cycles is Ae. aegypti L, which is limited to tropical regions, including sub-Saharan Africa and Southeast Asia. This disease usually develops 2e4 days after the mosquito bite. Patients display acute clinical signs of painful polyarthralgia and concomitant abrupt fever followed by skin rash, which is usually accompanied by headache and fatigue. These symptoms may persist for 3e7 days and then disappear after 2 weeks.4,5 However, arthralgia and myalgia may persist for months or years.6 CHIKV has a positive sense single stranded RNA genome of approximately 12,000 nucleotides in length, and it encodes four nonstructural (ns1e4) and three structural proteins (capsid, E1 and E2).7 Phylogenetic analysis revealed that there are three major CHIKV genotypes: (1) the West African genotype, distributed in West Africa; (2) the Central/East/South African (CESA) genotype, found outside West Africa; and (3) the Asian genotype, found mainly in Southeast Asia. In 2000, a CESA genotype was isolated from mosquito samples in India.8 Later, this virus strain was detected in local outbreaks in the Indian Ocean,9 India,10 Thailand,11 Italy,12 and Singapore.13 During this emerging epidemic, the responsible virus strain involved a mutation of changing alanine to valine at position 226 in the E1 envelope glycoprotein (E1/226V).9 This mutation largely increased the infectivity of Ae. albopictus.14,15 The responsible vector in the 2005e2006 CHIKV epidemics on Reunion Island and in the 2007 outbreak in Italy was apparently the Asian tiger mosquito, Ae. albopictus.12,16,17 This CHIKV vector species attracted more attention because of its wider distribution, including in temperate regions, and longer fly range (up to 600 m).18 This suggests that CHIKV transmission could occur in wider areas including tropical and temperate regions. In Taiwan, fever screenings at airports were launched as part of the active surveillance of travel-related diseases, such as dengue, malaria, and yellow fever, after the SARS outbreaks of 2003.19 Chikungunya was included in March 2006. Later, in November 2006, the first imported Chikungunya case from Singapore was detected.20 In October 2007, Chikungunya became a Category II notifiable communicable disease and physicians were required to report it within 24 hours. From October 2007 to September 2014, only 75 imported cases were detected. From these patients, three CHIKV strains, including the Asian, variant E1/226V, and nonvariant CESA genotypes, were isolated.21 As a result, infected travelers with high viremia coming/ returning from the endemic areas may generate a local transmission in Taiwan, such as in the case of dengue fever. Dengue viruses imported from Southeast Asian countries have caused local dengue outbreaks by Ae. aegypti and Ae. albopictus in Taiwan each year.22 The distribution of Ae. aegypti is geographically limited to the southern region of Taiwan, whereas Ae. albopictus is commonly found island
T.-H. Chen et al. wise below elevations of 1500 m above sea level.23 Therefore, the objective of this study was to evaluate the threat of CHIKVs through oral infection on vector mosquitoes, especially Ae. albopictus, which is commonly found throughout Taiwan.
Materials and methods Mosquito colony and maintenance Six mosquito colonies (1 Ae. aegypti and 5 Ae. albopictus) were used to test the susceptibility of CHIKV. Two laboratory colonies included the Tainan strain of Ae. aegypti, established in 1987, and the Minxiong strain of Ae. albopictus, established in 1997. Mosquito colonies had been maintained in an insectary at 20e30 C for an unknown number of generations. Four field colonies of Ae. albopictus were collected in 2011 from Bali District, New Taipei City in Northern Taiwan, Houli District, Taichung City in Central Taiwan, Taoyuan District, Kaohsiung City in Southern Taiwan, and Yuli Township, Hualien County in Eastern Taiwan (Fig. 1). The first generations of these field populations were used in the following experiments. Mosquito colonies were main tained in an insectary at 25 C with a photoperiod of 10:14 (light:dark) hours. Larvae were reared in a plastic pan (21 cm 14 cm 7 cm) containing 450 mL of deionized water. A sufficient amount of food (yeast powder and pig liver; 1:1 by weight) was provided daily. Adult mosquitoes were kept in an acrylic cage (29 cm 20 cm 20 cm) and were provided with a 10% sucrose solution.
Experimental oral infection Four-day-old female mosquitoes were deprived of sugar solution for 24 hours prior to the oral challenge. The feeding mixture was prepared by mixing equal parts of CHIKV-
Figure 1 Collection sites of Aedes albopictus mosquitoes in this study.
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Susceptibility of A. albopictus to Chikungunya virus infected C6/36 supernatant and human blood treated with anticoagulant (7 mL of human blood:18 mg of K2EDTA) (catalog number 367525, Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). The virus strains used in this study included Asian (CHIK/Indonesia/0811aTW/2008/FJ807891), and CESA (CHIK/Singapore/0611aTW/2006/FJ807896, and E1/226V CHIK/Malaysia/0901aTW/2009/FJ807895), which were isolated from blood specimens of the imported Chikungunya cases from Indonesia, Singapore, and Malaysia. All were harvested at concentrations of 106 plaque-forming units (PFU)/mL or 107 PFU/mL (determined by plaque assay).24 A high virus concentration of 1.98 106 PFU/mL was used for the following experiments and this concentration was close to the high virus load (ranging from 4.69 103 PFU/mL to 5.62 108 PFU/mL) of viremic patients.25 A total of 10e20 female mosquitoes were placed in a small paper cup (8 cm diameter 9.5 cm height; Yeong Hung Trading Co., LTD, Taichung, Taiwan) covered with fine nylon mesh and one drop (1 mL) of the feeding mixture was placed on the mesh of each cup. Mosquitoes were allowed to feed for 90 minutes with new blood drops being added every 30 minutes.26,27 The mosquitoes were then held in a growth chamber at 28 C and 75% relative humidity (RH). Cotton soaked in a 10% sugar solution was provided on the mesh. Mosquitoes were frozen at 0 day, 1 day, 2 day, 3 day, 4 day, 5 day, 6 day, 12 day, 18 day, and 24 day intervals until they died, and then they were stored at 80 C until being processed for virus detection.
Virus detection and titration A single mosquito was homogenized and clarified by centrifugation (9,560 g). Viral RNA (70 mL) was extracted from 140 mL of mosquito suspension using the QIAamp viral RNA mini kit (catalog number 52,906, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Amplification by reverse transcription polymerase chain reaction (RT-PCR) was performed using the Mx4000 quantitative PCR system (Stratagene, La Jolla, CA, USA). Samples were assayed in a 50 mL reaction mixture containing 10 mL of sample RNA and optimal concentrations of the primers using the QuantiTect SYBR Green RT-PCR kit (catalog number 204,243, Qiagen). Alphavirus-specific primers (AL-2: 50 -TAA TGC CAG AGC GTT TTC GCA-30 , AL-3: 50 -GTG GTG TCA AAC CCT ATC CA-30 , F-CHIK, and R-CHIK)20e22,24,28 were used for real-time RT-PCR. The former (AL-2 and AL-3) targeted a consensus region of the nonstructural protein 1 (nsp1) genes to detect all alphaviruses, and the products were expected to be 414 bp. The thermal profile consisted of a 30-minute reverse transcription step at 50 C and 15 minutes of Taq polymerase activation at 95 C, followed by 45 cycles of PCR (94 C for 15 seconds, annealing tempera ture 55 C for 30 seconds, 72 C for 20 seconds, and 77 C for 30 seconds). Following amplification, a melting curve analysis was performed to verify the correct product using its specific melting temperature. Additionally, serial 10-fold dilutions were undertaken of CHIKV (CESA strain) with an initial viral load of w106 PFU/mL (determined by plaque assay). Each dilution was added to 140 mL of one noninfected Ae. aegypti female mosquito suspension to estimate the virus titers in the orally-infected mosquitoes.
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Statistical analysis Statistical analyses were performed with STATISTICA 10 software (StatSoft, Inc., Tulsa, OK, USA). Transformations failed to make body titer data normally distributed. The rank tests (Mann-Whitney U test for two samples or KruskalWallis tests for multiple samples) were used to detect the difference in body titers among mosquito species, virus strains, and postinfection days.29,30
Results The fitness of CHIKV in laboratory-reared Aedes albopictus and Aedes aegypti First of all, the virus titer standard curve was performed by real time PCR to estimate the virus titers in the orallyinfected mosquitoes, and the linear regression of Ct value (Y) against log (viral load) (x) was Y Z 4.119 log(X) þ 36.77, with the R square value of 1.00. We evaluated the viral fitness of the Asian genotype CHIKV in both the laboratory-reared Ae. albopictus and Ae. aegypti in infectious blood at a titer of 1.98 106 PFU/mL. Only body titers of infected Ae. aegypti on 1 day post infection (dpi) (102.20) and 4 (102.76) were significantly (Z Z 3.84, df Z 1, p < 0.001; Z Z 2.25, df Z 1, p < 0.05) higher than those of infected Ae. albopcitus on dpi 1 (101.20) and 4 (101.52). The viral growth kinetics in Ae. aegypti females indicated that the number of viral genome copy slightly decreased, the mean (standard deviation) of viral genome at Day 0 was 103.08 0.17, but the viral copy number ranged from 102.12 0.23 to 102.76 0.88 at an early infectious stage (1e4 dpi; Fig. 2A). CHIKV genome amplification started increasing and peaked at 5 dpi (103.87 1.1). Then, it decreased over time at 8 dpi, 10 dpi, and 12 dpi (103.17 1.24, 103.14 1.66, and 102.55 1.64, respectively). The viral genome bounced back slightly at 18 dpi and 24 dpi (103.30 1.47 and 103.12 1.43, respectively). The viral growth curve in Ae. albopictus shows that the viral genome copy decreased (range from 101.22 1.06 to 102.28 0.48) at the first 4 days after infection (Fig. 2B). CHIKV increased in mosquitoes at 6 dpi (103.11 1.38) and peaked at 10 dpi (103.63 1.05). After 18 dpi, the viral genome largely decreased (103.06 0.86).
The growth curves of non-variant CHIKV and variant E1/226V strains in wild Aedes albopictus populations Next, we tested susceptibilities of the nonvariant and E1/ 226V variant CESA CHIKV in field-collected Ae. albopictus from Northern (Taipei), Central (Taichung), Eastern (Hualien), and Southern (Kaohsiung) Taiwan. For the nonvariant strain, body titers of four local mosquito strains were significantly (p < 0.05) different at 0 dpi, 1 dpi, 4 dpi, 12 dpi, 18 dpi, and 24 dpi. For the variant strain, body titers of these four local mosquito strains were significantly different (p < 0.05) at all dpi. The nonvariant viral copy numbers were increased at 1 dpi (104.18 0.41, 103.68 0.51) in the Taichung and Hualien strains, respectively (Fig. 3A
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 2 The growth curves of the Chikungunya virus (Asian genotype) in laboratory strains of Aedes aegypti (A) and Aedes albopictus (B) with initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection.
and C). In mosquitoes captured in Taipei and Kaohsiung, the viral copy number increased at 2 dpi (103.87 0.92 and 103.95 0.65; Fig. 3E and G). Since Day 4, viral genome amplification largely increased, with the viral genome number of 105.54 1.01, 105.67 0.86, and 104.76 0.64 in the Taichung, Hualien, and Taipei populations, respectively, and at 5 dpi in Kaohsiung captured mosquito (105.06 0.87). The maximum virus yields for Taichung, Hualien, Taipei, and Kaohsiung mosquitoes were 105.67 0.86 at 6 dpi, 106.27 0.40 at 12 dpi, 105.66 0.23 at 12 dpi, and 105.51 0.39 at 12 dpi, respectively. After mosquitoes were infected with the variant virus strain, the virus replicated immediately, and all the viral copy numbers were higher than that at 0 dpi in all Ae. albopictus mosquitoes at 1 dpi (viral RNA ranging from 103.93 0.57 to 104.71 0.46; Fig. 3B, D, F, and H). The viral growth curve reached its peak at 6 dpi in Taichung (106.09 0.35), 5 dpi in Hualien (106.23 0.52), and 4 dpi in Taipei (105.73 0.27) captured mosquitoes but peaked at 3 dpi (105.38 0.43) in Kaohsiung mosquitoes. We also evaluated susceptibilities of the nonvariant and E1/226V variant CHIKV in the laboratory-reared Ae. aegypti to provide a comparison with the results of Ae. albopictus. Body titers of the nonvariant and E1/226V variant were not significantly different (p > 0.10) on all dpi for Ae. aegypti. However, body titers of infected Ae. aegypti were significantly lower (p < 0.05) than those of some Ae. albopictus strains (Taichung and Hualien strains) for some dpi. The viral growth curve of nonvariant CHIKV decreased at 1 dpi (103.32 0.53) compared to 0 dpi (103.46 0.26; Fig. 4A). It started increasing at 2 dpi (104.25 1.39) and peaked at 4 dpi (105.38 1.04). Then, the number of viral RNA stayed steady until Day 24 pi. Additionally, the viral growth kinetics of the variant CHIKV indicated that the viral genome copy slightly increased (from 103.48 0.18 to 103.87 1.17) at the early infectious stage (0e2 dpi; Fig. 4B). It largely replicated at 3 dpi (105.08 0.63) and peaked at 5 dpi (105.62 0.64).
Discussion The E1/226V variant strains of CESA genotype have spread from Africa and India to other parts of the world and caused epidemics in Southeast Asian countries including Malaysia,
Thailand, Myanmar, Singapore, Indonesia, and China during 2008e2013. Although no local CHIKV outbreaks occurred in Taiwan, the introductions of the CHIKV viruses were identified from time to time through the imported cases. Therefore, we infected Ae. albopictus and Ae. aegypti with three imported CHIKV isolates in the laboratory. The virus susceptibility varied with virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5e6 dpi with the maximum virus yield, ranging from 103.63 to 103.87 at 5e10 dpi in both species. The variant CHIKV CESA virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 105.63 to 106.52 at 3e6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1e2 dpi with the maximum virus yield ranging from 105.51 to106.27 at 6e12 dpi. In Ae. aegypti, these viruses replicated at 1e2 dpi with the maximum yields at 4e5 dpi (range from 105.38 to 105.62). Therefore, we concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus and that the variant virus strain presents an even higher risk. In our study, susceptibility of the CHIKV varied by virus strain and mosquito species and strain, similar to studies of the CHIKV from American countries30 and the dengue virus.31 Some investigations have indicated that Ae. albopictus is more susceptible than Ae. aegypti to the variant type CHIKV,32 which is similar to three mosquito strains in our results. In the Kaohsiung mosquito strain, virus replication did not consistently occur. Previous studies have also shown that the variant strain CHIKV produced high viremia in monkeys, sufficient to infect Ae. albopictus and Ae. aegypti.33 In this study, E1/226V variant and nonvariant CHIKV infection rates were consistently higher in all strains of Ae. albopictus tested than in all strains of Ae. aegypti tested (Figs. 3 and 4). Additionally, the E1/226V CHIKV replicated at 1e2 dpi earlier than the nonvariant type virus and the Asian genotypes, indicating that the extrinsic incubation period may be shorter and indicating efficient transmission to other hosts. Determination of vector competence of mosquito populations is a key parameter in evaluating the risk of CHIKV transmission and spread. Vector competence is determined by the preference of mosquito species on human blood and virus replication in mosquito vectors including viral load and speed of replication. Both Ae. aegypt and Ae. albopictus
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 3 At different days after oral infection, viral RNA was measured by real time reverse transcription polymerase chain reaction. The growth curves of non-variant CHIKV (Central/East/South African genotype) in Aedes albopictus collected in Taichung (A), Hualien (C), Taipei (E), and Kaohsiung (G), and E1/226V variant in Aedes albopictus collected in Taichung (B), Hualien (D), Taipei (F) and Kaohsiung (H) with an initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection. Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 4 The growth curves of nonvariant CHIKV (Central/East/South African genotype) (A) and E1/226V variant (B) and in a Tainan Aedes aegypti laboratory strain with an initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection.
have the preference of feeding on human blood.34,35 The speed of replication represents the duration of the extrinsic incubation period. No papers were available on the correlation of the viral load in mosquitoes to the actual occurrence of transmission and/or outbreaks in vivo. However, high viral loads of CHIKV were commonly detected both in the field-collected mosquito samples (3.26 104 copies/ mL) and human patient samples on Day 1 of fever onset (8.57 106 copies/mL) during outbreaks, such as a Chikungunya fever outbreak in 2008.36 Therefore, the higher viral load and speed of replication in mosquitoes was considered the higher risk of the disease in this paper. In Taiwan, the dengue virus is introduced from abroad by infected travelers during the early summer each year. Later, the virus is vectored locally by mosquitoes and usually disappears during the dry and cold winter season. The vector responsible for the outbreaks is Ae. aegypti in Southern Taiwan and Ae. albopictus in the areas where Ae. aegypti is absent. Although no local mosquito-transmitted cases of Chikungunya in Taiwan have been reported, the possibility cannot be excluded due to the similar symptoms of these two diseases.37 Historical reports indicate that some epidemics caused by the dengue virus may also have been caused by CHIKV.38,39 In Taiwan, the CHIKV virus strains isolated from imported cases include the Asian, E1226V variant, and nonvariant CESA genotypes.21 Therefore, it is very likely that this pattern of dengue transmission could also occur with CHIKV. Mosquito control is the sole available method for reducing transmission of Chikungunya, and no vaccines are available. Control of this disease, as with dengue, is highly dependent on source reduction to reduce vector density to prevent the occurrence of this disease.1,2 Fortunately, this control method is working for CHIKV as well as for dengue.35,36 Finally, we conclude that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype, and that the variant virus strain presents an even higher risk. Dengue vector surveillance in Taiwan was conducted by local health bureaus and population density is represented as adult index (the number of Aedes adults, Ae. aegypti or Ae. albopictus per 100 houses) or traditional Stegomyia indices, house index (the percentage of houses infested with Aedes immatures), Breteaux index (the number of
containers infested with Aedes immatures per 100 houses), and container index (the percentage of water-holding containers infested with Aedes immatures). The dengue control strategies include source reduction and insecticide sprays, in which the application frequencies of insecticides were based on extrinsic incubation period in mosquitoes.40 Therefore, the rapid replication of CHKV in our study will affect the frequencies of insecticide sprays from a week in dengue control to 4 days to battle the Chikungunya.
Acknowledgments This study was supported by scientific research grants from the Centers for Disease Control, Taiwan in 2009e2011 (DOH98-DC-2013, DOH99-DC-2030, and DOH100-DC-2018).
References 1. Lumsden WH. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952e53. II. General description and epidemiology. Trans R Soc Trop Med Hyg 1955;49:33e57. 2. Powers AM, Logue CH. Changing patterns of Chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol 2007;88: 2363e77. 3. Robinson MC. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952e53. I. Clinical features. Trans R Soc Trop Med Hyg 1955;49:28e32. 4. Brighton SW, Prozesky OW, de la Harpe AL. Chikungunya virus infection. A retrospective study of 107 cases. S Afr Med J 1983; 63:313e5. 5. Jaffar-Bandjee MC, Ramful D, Gauzere BA, Hoarau JJ, Krejbich-Trotot P, Robin S, et al. Emergence and clinical insights into the pathology of Chikungunya virus infection. Expert Rev Anti Infect Ther 2010;8:987e96. 6. Dupuis-Maguiraga L, Noret M, Brun S, Le Grand R, Gras G, Roques P. Chikungunya disease: infection-associated markers from the acute to the chronic phase of arbovirus-induced arthralgia. PLoS Negl Trop Dis 2012;6:e1446. 7. Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 1994;58:491e562. 8. Yergolkar PN, Tandale BV, Arankalle VA, Sathe PS, Sudeep AB, Gandhe SS, et al. Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis 2006;12:1580e3.
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Susceptibility of A. albopictus to Chikungunya virus 9. Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, et al. Genome microevolution of Chikungunya viruses causing the Indian Ocean outbreak. PLoS Med 2006;3: e263. 10. Arankalle VA, Shrivastava S, Cherian S, Gunjikar RS, Walimbe AM, Jadhav SM, et al. Genetic divergence of Chikungunya viruses in India (1963e2006) with special reference to the 2005e2006 explosive epidemic. J Gen Virol 2007;88: 1967e76. 11. Rianthavorn P, Prianantathavorn K, Wuttirattanakowit N, Theamboonlers A, Poovorawan Y. An outbreak of Chikungunya in southern Thailand from 2008 to 2009 caused by African strains with A226V mutation. Intern J Infect Dis 2010;14(Suppl 3):e161e5. 12. Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, et al. Infection with Chikungunya virus in Italy: an outbreak in a temperate region. Lancet 2007;370:1840e6. 13. Ng LC, Tan LK, Tan CH, Tan SS, Hapuarachchi HC, Pok KY, et al. Entomologic and virologic investigation of Chikungunya, Singapore. Emerg Infect Dis 2009;15:1243e9. 14. de Lamballerie X, Leroy E, Charrel RN, Ttsetsarkin K, Higgs S, Gould EA. Chikungunya virus adapts to tiger mosquito via evolutionary convergence: a sign of things to come? Virol J 2008;5:33. 15. Kielian M. Structural biology: An alphavirus puzzle solved. Nature 2010;468:645e6. 16. Enserink M. Infectious diseases. Massive outbreak draws fresh attention to little-known virus. Science 2006;311:1085. 17. Reiter P, Fontenille D, Paupy C. Aedes albopictus as an epidemic vector of Chikungunya virus: another emerging problem? Lancet Infect Dis 2006;6:463e4. 18. Niebylski ML, Craig Jr GB. Dispersal and survival of Aedes albopictus at a scrap tire yard in Missouri. J Am Mosq Control Assoc 1994;10:339e43. 19. Shu PY, Chien LJ, Chang SF, Su CL, Kuo YC, Liao TL, et al. Fever screening at airports and imported dengue. Emerg Infect Dis 2005;11:460e2. 20. Shu PY, Yang CF, Su CL, Chen CY, Chang SF, Tsai KH, et al. Two imported Chikungunya cases, Taiwan. Emerg Infect Dis 2008; 14:1326e7. 21. Huang JH, Yang CF, Su CL, Chang SF, Cheng CH, Yu SK, et al. Imported Chikungunya virus strains, Taiwan, 2006e2009. Emerg Infect Dis 2009;15:1854e6. 22. Chang SF, Huang JH, Shu PY. Characteristics of dengue epidemics in Taiwan. J Formos Med Assoc 2012;111:297e9. 23. Lin C, Wang CY, Teng HJ. The study of dengue vector distribution in Taiwan from 2009 to 2011. Taiwan Epidemiol Bull 2014;30:304e10. 24. Shu PY, Chang SF, Kuo YC, Yueh YY, Chien LJ, Sue CL, et al. Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus. J Clin Microbiol 2003;41:2408e16. 25. Chow A, Her Z, Ong EKS, Chen JM, Dimatatac F, Kwek DJC, et al. Persistent arthralgia induced by Chikungunya virus infection is associated with interleukin-6 and granulocyte
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macrophage colony-stimulating factor. J Infect Dis 2011;203: 149e57. Dubrulle M, Mousson L, Moutailler S, Vazeille M, Failloux A-B. Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection. PLoS One 2009;4:e5895. Yang CF, Shu PY, Chen CF, Su CL, Teng HJ, Lu LC, et al. Screening of mosquito infections using SYBR Green I-based real time RT-PCR with group primers of Flavivirus and Alphavirus in Taiwan. J Virol Methods 2010;168:147e51. Pastorino B, Bessaud M, Grandadam M, Murri S, Tolou HJ, Peyrefitte CN. Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses. J Virol Methods 2005;124:65e71. Reiskind MH, Pesko K, Westbrook CJ, Mores CN. Susceptibility of Florida mosquitoes to infection with Chikungunya virus. Am J Trop Med Hyg 2008;78:422e5. Vega-Rue A, Zouzche K, Girod R, Failloux AB, Lourenco-deOliveira R. High level of vector competence of Aedes aegypti and Aedes albopictus from ten American countries as a crucial factor in the spread of Chikungunya virus. J Virol 2014;88: 6294e306. Tesh RB, Gubler DJ, Rosen L. Variation among geographic strains of Aedes albopictus in susceptibility to infection with Chikungunya virus. Am J Trop Med Hyg 1976;25:326e35. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation in Chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 2007;3:e201. Turell MJ, Beaman JR, Tammariello RF. Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to Chikungunya virus. J Med Entomol 1992;29:49e53. Harrington LC, Edman JD, Scott TW. Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood? J Med Entomol 2001;38:411e22. Kamgang B, Nchoutpouen E, Simard F, Paupy C. Notes on the blood-feeding behavior of Aedes albopictus (Diptera: Culicidae) in Cameroon. Parasit Vectors 2012;5:57. Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S. Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples. Trop Biomed 2010;27:611e23. Nimmannitya S, Halstead SB, Cohen SN, Margiotta MR. Dengue and Chikungunya virus infection in man in Thailand, 1962-1964. I. Observations on hospitalized patients with hemorrhagic fever. Am J Trop Med Hyg 1969;18:954e71. Carey DE. Chikungunya and dengue: a case of mistaken identity? J Hist Med Allied Sci 1971;26:243e62. Halstead SB, Nimmannitya S, Margiotta MR. Dengue d Chikungunya virus infection in man in Thailand, 1962-1964. II. Observations on disease in outpatients. Am J Trop Med Hyg 1969;18:972e83. Teng HJ, Chen TJ, Tsai SF, Lin CP, Chiou HY, Lin MC, et al. Emergency vector control in a DENV-2 outbreak in 2002 in Pingtung City, Pingtung County, Taiwan. Jap J Infect Dis 2007; 2007(60):271e9.
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Journal of the Formosan Medical Association (2015) xx, 1e7
Available online at www.sciencedirect.com
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ORIGINAL ARTICLE
Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan Tien-Huang Chen, Shu-Wan Jian, Chih-Yuan Wang, Cheo Lin, Pei-Feng Wang, Chien-Ling Su, Hwa-Jen Teng*, Pei-Yun Shu, Ho-Sheng Wu Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Number 161, Kunyang Street, Taipei 11561, Taiwan, ROC Received 17 August 2014; received in revised form 3 December 2014; accepted 26 December 2014
KEYWORDS Aedes aegypti; Aedes albopictus; Chikungunya virus; mosquito infection; Taiwan
Background/purpose: An E1/226V variant Chikungunya virus (CHIKV) efficiently transmitted by Aedes albopictus to humans poses a significant threat to public health for those areas with the presence of Aedes albopictus, including Taiwan. Methods: We infected three imported CHIKV isolates including the E1/226V variant with Ae. albopictus and Aedes aegypti in the laboratory to understand the disease risk. Viral RNA was measured by real time reverse transcription polymerase chain reaction. Results: The viral susceptibility varied by virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5e6 days post infection (dpi) with the maximum virus yield, ranging from 103.63 to 103.87 at 5e10 dpi in both species. The variant CHIKV Central/ East/South African (CESA) virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 105.63 to 106.52 at 3e6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1e2 dpi with the maximum virus yield ranging from 105.51 to106.27 at 6e12 dpi. In Ae. aegypti, these viruses replicated at 1e2 dpi, with maximum yields at 4 e5 dpi (range from 105.38 to 105.62). Conclusion: We concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype and that the E1/226V variant virus strain presents an even higher risk. Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Conflicts of interest: The authors have no conflicts of interest relevant to this article. * Corresponding author. Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Number 161, Kunyang Street, Taipei 11561, Taiwan, ROC. E-mail address: [email protected] (H.-J. Teng). http://dx.doi.org/10.1016/j.jfma.2014.12.002 0929-6646/Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
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Introduction Chikungunya virus (CHIKV) is a member of Alphavirus (Togaviridae) and is transmitted to human beings by Aedes mosquitoes. It was first isolated from a patient in Tanzania in 1953.1 Later, it caused numerous epidemic cases in Africa and Asia.2,3 This virus has urban and sylvatic transmission cycles, which have their own vector species. The main vector species responsible for urban transmission cycles is Ae. aegypti L, which is limited to tropical regions, including sub-Saharan Africa and Southeast Asia. This disease usually develops 2e4 days after the mosquito bite. Patients display acute clinical signs of painful polyarthralgia and concomitant abrupt fever followed by skin rash, which is usually accompanied by headache and fatigue. These symptoms may persist for 3e7 days and then disappear after 2 weeks.4,5 However, arthralgia and myalgia may persist for months or years.6 CHIKV has a positive sense single stranded RNA genome of approximately 12,000 nucleotides in length, and it encodes four nonstructural (ns1e4) and three structural proteins (capsid, E1 and E2).7 Phylogenetic analysis revealed that there are three major CHIKV genotypes: (1) the West African genotype, distributed in West Africa; (2) the Central/East/South African (CESA) genotype, found outside West Africa; and (3) the Asian genotype, found mainly in Southeast Asia. In 2000, a CESA genotype was isolated from mosquito samples in India.8 Later, this virus strain was detected in local outbreaks in the Indian Ocean,9 India,10 Thailand,11 Italy,12 and Singapore.13 During this emerging epidemic, the responsible virus strain involved a mutation of changing alanine to valine at position 226 in the E1 envelope glycoprotein (E1/226V).9 This mutation largely increased the infectivity of Ae. albopictus.14,15 The responsible vector in the 2005e2006 CHIKV epidemics on Reunion Island and in the 2007 outbreak in Italy was apparently the Asian tiger mosquito, Ae. albopictus.12,16,17 This CHIKV vector species attracted more attention because of its wider distribution, including in temperate regions, and longer fly range (up to 600 m).18 This suggests that CHIKV transmission could occur in wider areas including tropical and temperate regions. In Taiwan, fever screenings at airports were launched as part of the active surveillance of travel-related diseases, such as dengue, malaria, and yellow fever, after the SARS outbreaks of 2003.19 Chikungunya was included in March 2006. Later, in November 2006, the first imported Chikungunya case from Singapore was detected.20 In October 2007, Chikungunya became a Category II notifiable communicable disease and physicians were required to report it within 24 hours. From October 2007 to September 2014, only 75 imported cases were detected. From these patients, three CHIKV strains, including the Asian, variant E1/226V, and nonvariant CESA genotypes, were isolated.21 As a result, infected travelers with high viremia coming/ returning from the endemic areas may generate a local transmission in Taiwan, such as in the case of dengue fever. Dengue viruses imported from Southeast Asian countries have caused local dengue outbreaks by Ae. aegypti and Ae. albopictus in Taiwan each year.22 The distribution of Ae. aegypti is geographically limited to the southern region of Taiwan, whereas Ae. albopictus is commonly found island
T.-H. Chen et al. wise below elevations of 1500 m above sea level.23 Therefore, the objective of this study was to evaluate the threat of CHIKVs through oral infection on vector mosquitoes, especially Ae. albopictus, which is commonly found throughout Taiwan.
Materials and methods Mosquito colony and maintenance Six mosquito colonies (1 Ae. aegypti and 5 Ae. albopictus) were used to test the susceptibility of CHIKV. Two laboratory colonies included the Tainan strain of Ae. aegypti, established in 1987, and the Minxiong strain of Ae. albopictus, established in 1997. Mosquito colonies had been maintained in an insectary at 20e30 C for an unknown number of generations. Four field colonies of Ae. albopictus were collected in 2011 from Bali District, New Taipei City in Northern Taiwan, Houli District, Taichung City in Central Taiwan, Taoyuan District, Kaohsiung City in Southern Taiwan, and Yuli Township, Hualien County in Eastern Taiwan (Fig. 1). The first generations of these field populations were used in the following experiments. Mosquito colonies were main tained in an insectary at 25 C with a photoperiod of 10:14 (light:dark) hours. Larvae were reared in a plastic pan (21 cm 14 cm 7 cm) containing 450 mL of deionized water. A sufficient amount of food (yeast powder and pig liver; 1:1 by weight) was provided daily. Adult mosquitoes were kept in an acrylic cage (29 cm 20 cm 20 cm) and were provided with a 10% sucrose solution.
Experimental oral infection Four-day-old female mosquitoes were deprived of sugar solution for 24 hours prior to the oral challenge. The feeding mixture was prepared by mixing equal parts of CHIKV-
Figure 1 Collection sites of Aedes albopictus mosquitoes in this study.
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Susceptibility of A. albopictus to Chikungunya virus infected C6/36 supernatant and human blood treated with anticoagulant (7 mL of human blood:18 mg of K2EDTA) (catalog number 367525, Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). The virus strains used in this study included Asian (CHIK/Indonesia/0811aTW/2008/FJ807891), and CESA (CHIK/Singapore/0611aTW/2006/FJ807896, and E1/226V CHIK/Malaysia/0901aTW/2009/FJ807895), which were isolated from blood specimens of the imported Chikungunya cases from Indonesia, Singapore, and Malaysia. All were harvested at concentrations of 106 plaque-forming units (PFU)/mL or 107 PFU/mL (determined by plaque assay).24 A high virus concentration of 1.98 106 PFU/mL was used for the following experiments and this concentration was close to the high virus load (ranging from 4.69 103 PFU/mL to 5.62 108 PFU/mL) of viremic patients.25 A total of 10e20 female mosquitoes were placed in a small paper cup (8 cm diameter 9.5 cm height; Yeong Hung Trading Co., LTD, Taichung, Taiwan) covered with fine nylon mesh and one drop (1 mL) of the feeding mixture was placed on the mesh of each cup. Mosquitoes were allowed to feed for 90 minutes with new blood drops being added every 30 minutes.26,27 The mosquitoes were then held in a growth chamber at 28 C and 75% relative humidity (RH). Cotton soaked in a 10% sugar solution was provided on the mesh. Mosquitoes were frozen at 0 day, 1 day, 2 day, 3 day, 4 day, 5 day, 6 day, 12 day, 18 day, and 24 day intervals until they died, and then they were stored at 80 C until being processed for virus detection.
Virus detection and titration A single mosquito was homogenized and clarified by centrifugation (9,560 g). Viral RNA (70 mL) was extracted from 140 mL of mosquito suspension using the QIAamp viral RNA mini kit (catalog number 52,906, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Amplification by reverse transcription polymerase chain reaction (RT-PCR) was performed using the Mx4000 quantitative PCR system (Stratagene, La Jolla, CA, USA). Samples were assayed in a 50 mL reaction mixture containing 10 mL of sample RNA and optimal concentrations of the primers using the QuantiTect SYBR Green RT-PCR kit (catalog number 204,243, Qiagen). Alphavirus-specific primers (AL-2: 50 -TAA TGC CAG AGC GTT TTC GCA-30 , AL-3: 50 -GTG GTG TCA AAC CCT ATC CA-30 , F-CHIK, and R-CHIK)20e22,24,28 were used for real-time RT-PCR. The former (AL-2 and AL-3) targeted a consensus region of the nonstructural protein 1 (nsp1) genes to detect all alphaviruses, and the products were expected to be 414 bp. The thermal profile consisted of a 30-minute reverse transcription step at 50 C and 15 minutes of Taq polymerase activation at 95 C, followed by 45 cycles of PCR (94 C for 15 seconds, annealing tempera ture 55 C for 30 seconds, 72 C for 20 seconds, and 77 C for 30 seconds). Following amplification, a melting curve analysis was performed to verify the correct product using its specific melting temperature. Additionally, serial 10-fold dilutions were undertaken of CHIKV (CESA strain) with an initial viral load of w106 PFU/mL (determined by plaque assay). Each dilution was added to 140 mL of one noninfected Ae. aegypti female mosquito suspension to estimate the virus titers in the orally-infected mosquitoes.
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Statistical analysis Statistical analyses were performed with STATISTICA 10 software (StatSoft, Inc., Tulsa, OK, USA). Transformations failed to make body titer data normally distributed. The rank tests (Mann-Whitney U test for two samples or KruskalWallis tests for multiple samples) were used to detect the difference in body titers among mosquito species, virus strains, and postinfection days.29,30
Results The fitness of CHIKV in laboratory-reared Aedes albopictus and Aedes aegypti First of all, the virus titer standard curve was performed by real time PCR to estimate the virus titers in the orallyinfected mosquitoes, and the linear regression of Ct value (Y) against log (viral load) (x) was Y Z 4.119 log(X) þ 36.77, with the R square value of 1.00. We evaluated the viral fitness of the Asian genotype CHIKV in both the laboratory-reared Ae. albopictus and Ae. aegypti in infectious blood at a titer of 1.98 106 PFU/mL. Only body titers of infected Ae. aegypti on 1 day post infection (dpi) (102.20) and 4 (102.76) were significantly (Z Z 3.84, df Z 1, p < 0.001; Z Z 2.25, df Z 1, p < 0.05) higher than those of infected Ae. albopcitus on dpi 1 (101.20) and 4 (101.52). The viral growth kinetics in Ae. aegypti females indicated that the number of viral genome copy slightly decreased, the mean (standard deviation) of viral genome at Day 0 was 103.08 0.17, but the viral copy number ranged from 102.12 0.23 to 102.76 0.88 at an early infectious stage (1e4 dpi; Fig. 2A). CHIKV genome amplification started increasing and peaked at 5 dpi (103.87 1.1). Then, it decreased over time at 8 dpi, 10 dpi, and 12 dpi (103.17 1.24, 103.14 1.66, and 102.55 1.64, respectively). The viral genome bounced back slightly at 18 dpi and 24 dpi (103.30 1.47 and 103.12 1.43, respectively). The viral growth curve in Ae. albopictus shows that the viral genome copy decreased (range from 101.22 1.06 to 102.28 0.48) at the first 4 days after infection (Fig. 2B). CHIKV increased in mosquitoes at 6 dpi (103.11 1.38) and peaked at 10 dpi (103.63 1.05). After 18 dpi, the viral genome largely decreased (103.06 0.86).
The growth curves of non-variant CHIKV and variant E1/226V strains in wild Aedes albopictus populations Next, we tested susceptibilities of the nonvariant and E1/ 226V variant CESA CHIKV in field-collected Ae. albopictus from Northern (Taipei), Central (Taichung), Eastern (Hualien), and Southern (Kaohsiung) Taiwan. For the nonvariant strain, body titers of four local mosquito strains were significantly (p < 0.05) different at 0 dpi, 1 dpi, 4 dpi, 12 dpi, 18 dpi, and 24 dpi. For the variant strain, body titers of these four local mosquito strains were significantly different (p < 0.05) at all dpi. The nonvariant viral copy numbers were increased at 1 dpi (104.18 0.41, 103.68 0.51) in the Taichung and Hualien strains, respectively (Fig. 3A
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 2 The growth curves of the Chikungunya virus (Asian genotype) in laboratory strains of Aedes aegypti (A) and Aedes albopictus (B) with initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection.
and C). In mosquitoes captured in Taipei and Kaohsiung, the viral copy number increased at 2 dpi (103.87 0.92 and 103.95 0.65; Fig. 3E and G). Since Day 4, viral genome amplification largely increased, with the viral genome number of 105.54 1.01, 105.67 0.86, and 104.76 0.64 in the Taichung, Hualien, and Taipei populations, respectively, and at 5 dpi in Kaohsiung captured mosquito (105.06 0.87). The maximum virus yields for Taichung, Hualien, Taipei, and Kaohsiung mosquitoes were 105.67 0.86 at 6 dpi, 106.27 0.40 at 12 dpi, 105.66 0.23 at 12 dpi, and 105.51 0.39 at 12 dpi, respectively. After mosquitoes were infected with the variant virus strain, the virus replicated immediately, and all the viral copy numbers were higher than that at 0 dpi in all Ae. albopictus mosquitoes at 1 dpi (viral RNA ranging from 103.93 0.57 to 104.71 0.46; Fig. 3B, D, F, and H). The viral growth curve reached its peak at 6 dpi in Taichung (106.09 0.35), 5 dpi in Hualien (106.23 0.52), and 4 dpi in Taipei (105.73 0.27) captured mosquitoes but peaked at 3 dpi (105.38 0.43) in Kaohsiung mosquitoes. We also evaluated susceptibilities of the nonvariant and E1/226V variant CHIKV in the laboratory-reared Ae. aegypti to provide a comparison with the results of Ae. albopictus. Body titers of the nonvariant and E1/226V variant were not significantly different (p > 0.10) on all dpi for Ae. aegypti. However, body titers of infected Ae. aegypti were significantly lower (p < 0.05) than those of some Ae. albopictus strains (Taichung and Hualien strains) for some dpi. The viral growth curve of nonvariant CHIKV decreased at 1 dpi (103.32 0.53) compared to 0 dpi (103.46 0.26; Fig. 4A). It started increasing at 2 dpi (104.25 1.39) and peaked at 4 dpi (105.38 1.04). Then, the number of viral RNA stayed steady until Day 24 pi. Additionally, the viral growth kinetics of the variant CHIKV indicated that the viral genome copy slightly increased (from 103.48 0.18 to 103.87 1.17) at the early infectious stage (0e2 dpi; Fig. 4B). It largely replicated at 3 dpi (105.08 0.63) and peaked at 5 dpi (105.62 0.64).
Discussion The E1/226V variant strains of CESA genotype have spread from Africa and India to other parts of the world and caused epidemics in Southeast Asian countries including Malaysia,
Thailand, Myanmar, Singapore, Indonesia, and China during 2008e2013. Although no local CHIKV outbreaks occurred in Taiwan, the introductions of the CHIKV viruses were identified from time to time through the imported cases. Therefore, we infected Ae. albopictus and Ae. aegypti with three imported CHIKV isolates in the laboratory. The virus susceptibility varied with virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5e6 dpi with the maximum virus yield, ranging from 103.63 to 103.87 at 5e10 dpi in both species. The variant CHIKV CESA virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 105.63 to 106.52 at 3e6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1e2 dpi with the maximum virus yield ranging from 105.51 to106.27 at 6e12 dpi. In Ae. aegypti, these viruses replicated at 1e2 dpi with the maximum yields at 4e5 dpi (range from 105.38 to 105.62). Therefore, we concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus and that the variant virus strain presents an even higher risk. In our study, susceptibility of the CHIKV varied by virus strain and mosquito species and strain, similar to studies of the CHIKV from American countries30 and the dengue virus.31 Some investigations have indicated that Ae. albopictus is more susceptible than Ae. aegypti to the variant type CHIKV,32 which is similar to three mosquito strains in our results. In the Kaohsiung mosquito strain, virus replication did not consistently occur. Previous studies have also shown that the variant strain CHIKV produced high viremia in monkeys, sufficient to infect Ae. albopictus and Ae. aegypti.33 In this study, E1/226V variant and nonvariant CHIKV infection rates were consistently higher in all strains of Ae. albopictus tested than in all strains of Ae. aegypti tested (Figs. 3 and 4). Additionally, the E1/226V CHIKV replicated at 1e2 dpi earlier than the nonvariant type virus and the Asian genotypes, indicating that the extrinsic incubation period may be shorter and indicating efficient transmission to other hosts. Determination of vector competence of mosquito populations is a key parameter in evaluating the risk of CHIKV transmission and spread. Vector competence is determined by the preference of mosquito species on human blood and virus replication in mosquito vectors including viral load and speed of replication. Both Ae. aegypt and Ae. albopictus
Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 3 At different days after oral infection, viral RNA was measured by real time reverse transcription polymerase chain reaction. The growth curves of non-variant CHIKV (Central/East/South African genotype) in Aedes albopictus collected in Taichung (A), Hualien (C), Taipei (E), and Kaohsiung (G), and E1/226V variant in Aedes albopictus collected in Taichung (B), Hualien (D), Taipei (F) and Kaohsiung (H) with an initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection. Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
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Figure 4 The growth curves of nonvariant CHIKV (Central/East/South African genotype) (A) and E1/226V variant (B) and in a Tainan Aedes aegypti laboratory strain with an initial viral load of 1.98 106 plaque-forming units (PFU)/mL (determined by plaque assay). dpi Z days post infection.
have the preference of feeding on human blood.34,35 The speed of replication represents the duration of the extrinsic incubation period. No papers were available on the correlation of the viral load in mosquitoes to the actual occurrence of transmission and/or outbreaks in vivo. However, high viral loads of CHIKV were commonly detected both in the field-collected mosquito samples (3.26 104 copies/ mL) and human patient samples on Day 1 of fever onset (8.57 106 copies/mL) during outbreaks, such as a Chikungunya fever outbreak in 2008.36 Therefore, the higher viral load and speed of replication in mosquitoes was considered the higher risk of the disease in this paper. In Taiwan, the dengue virus is introduced from abroad by infected travelers during the early summer each year. Later, the virus is vectored locally by mosquitoes and usually disappears during the dry and cold winter season. The vector responsible for the outbreaks is Ae. aegypti in Southern Taiwan and Ae. albopictus in the areas where Ae. aegypti is absent. Although no local mosquito-transmitted cases of Chikungunya in Taiwan have been reported, the possibility cannot be excluded due to the similar symptoms of these two diseases.37 Historical reports indicate that some epidemics caused by the dengue virus may also have been caused by CHIKV.38,39 In Taiwan, the CHIKV virus strains isolated from imported cases include the Asian, E1226V variant, and nonvariant CESA genotypes.21 Therefore, it is very likely that this pattern of dengue transmission could also occur with CHIKV. Mosquito control is the sole available method for reducing transmission of Chikungunya, and no vaccines are available. Control of this disease, as with dengue, is highly dependent on source reduction to reduce vector density to prevent the occurrence of this disease.1,2 Fortunately, this control method is working for CHIKV as well as for dengue.35,36 Finally, we conclude that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype, and that the variant virus strain presents an even higher risk. Dengue vector surveillance in Taiwan was conducted by local health bureaus and population density is represented as adult index (the number of Aedes adults, Ae. aegypti or Ae. albopictus per 100 houses) or traditional Stegomyia indices, house index (the percentage of houses infested with Aedes immatures), Breteaux index (the number of
containers infested with Aedes immatures per 100 houses), and container index (the percentage of water-holding containers infested with Aedes immatures). The dengue control strategies include source reduction and insecticide sprays, in which the application frequencies of insecticides were based on extrinsic incubation period in mosquitoes.40 Therefore, the rapid replication of CHKV in our study will affect the frequencies of insecticide sprays from a week in dengue control to 4 days to battle the Chikungunya.
Acknowledgments This study was supported by scientific research grants from the Centers for Disease Control, Taiwan in 2009e2011 (DOH98-DC-2013, DOH99-DC-2030, and DOH100-DC-2018).
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Please cite this article in press as: Chen T-H, et al., Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/ j.jfma.2014.12.002
