Journal of the Formosan Medical Association (2015) 114, 546e552

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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 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, 11561, Taiwan, ROC

Received 17 August 2014; received in revised form 3 December 2014; accepted 26 December 2014

KEYWORDS Background/purpose: An E1/226V variant Chikungunya virus (CHIKV) efficiently transmitted by Aedes aegypti; Aedes albopictus to humans poses a significant threat to public health for those areas with the Aedes albopictus; presence of Aedes albopictus, including Taiwan. Chikungunya virus; Methods: We infected three imported CHIKV isolates including the E1/226V variant with Ae. mosquito infection; albopictus and Aedes aegypti in the laboratory to understand the disease risk. Viral RNA was Taiwan 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 3e6dpiinAe. 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. Susceptibility of A. albopictus to Chikungunya virus 547

Introduction wise below elevations of 1500 m above sea level.23 There- fore, the objective of this study was to evaluate the threat Chikungunya virus (CHIKV) is a member of Alphavirus of CHIKVs through oral infection on vector mosquitoes, (Togaviridae) and is transmitted to human beings by Aedes especially Ae. albopictus, which is commonly found mosquitoes. It was first isolated from a patient in Tanzania throughout Taiwan. in 1953.1 Later, it caused numerous epidemic cases in Africa 2,3 and Asia. This virus has urban and sylvatic transmission Materials and methods cycles, which have their own vector species. The main vector species responsible for urban transmission cycles is Mosquito colony and maintenance 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 Six mosquito colonies (1 Ae. aegypti and 5 Ae. albopictus) acute clinical signs of painful polyarthralgia and concomi- were used to test the susceptibility of CHIKV. Two laboratory tant abrupt fever followed by skin rash, which is usually colonies included the strain of Ae. aegypti, estab- accompanied by headache and fatigue. These symptoms lished in 1987, and the Minxiong strain of Ae. albopictus, may persist for 3e7 days and then disappear after 2 established in 1997. Mosquito colonies had been maintained e weeks.4,5 However, arthralgia and myalgia may persist for in an insectary at 20 30 C for an unknown number of gen- months or years.6 erations. Four field colonies of Ae. albopictus were collected CHIKV has a positive sense single stranded RNA genome in 2011 from Bali , in Northern of approximately 12,000 nucleotides in length, and it en- Taiwan, , City in Central Taiwan, codes four nonstructural (ns1e4) and three structural pro- Taoyuan District, City in Southern Taiwan, and teins (capsid, E1 and E2).7 Phylogenetic analysis revealed Yuli , Hualien County in Eastern Taiwan (Fig. 1). that there are three major CHIKV genotypes: (1) the West The first generations of these field populations were used in the following experiments. Mosquito colonies were main- African genotype, distributed in West Africa; (2) the Cen- tral/East/South African (CESA) genotype, found outside tained in an insectary at 25 C with a photoperiod of 10:14 West Africa; and (3) the Asian genotype, found mainly in (light:dark) hours. Larvae were reared in a plastic pan Southeast Asia. In 2000, a CESA genotype was isolated from (21 cm 14 cm 7 cm) containing 450 mL of deionized mosquito samples in India.8 Later, this virus strain was water. A sufficient amount of food (yeast powder and pig detected in local outbreaks in the Indian Ocean,9 India,10 liver; 1:1 by weight) was provided daily. Adult mosquitoes Thailand,11 Italy,12 and Singapore.13 During this emerging were kept in an acrylic cage (29 cm 20 cm 20 cm) and epidemic, the responsible virus strain involved a mutation were provided with a 10% sucrose solution. of changing alanine to valine at position 226 in the E1 en- velope glycoprotein (E1/226V).9 This mutation largely Experimental oral infection increased the infectivity of Ae. albopictus.14,15 The responsible vector in the 2005e2006 CHIKV epidemics on Four-day-old female mosquitoes were deprived of sugar Reunion Island and in the 2007 outbreak in Italy was solution for 24 hours prior to the oral challenge. The feeding apparently the Asian tiger mosquito, Ae. albopictus.12,16,17 mixture was prepared by mixing equal parts of CHIKV- 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 Chi- kungunya 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 Figure 1 Collection sites of Aedes albopictus mosquitoes in Taiwan, whereas Ae. albopictus is commonly found island this study. 548 T.-H. Chen et al. infected C6/36 supernatant and human blood treated with Statistical analysis anticoagulant (7 mL of human blood:18 mg of K2EDTA) (catalog number 367525, Becton, Dickinson, and Company, Statistical analyses were performed with STATISTICA 10 Franklin Lakes, NJ, USA). The virus strains used in this study software (StatSoft, Inc., Tulsa, OK, USA). Transformations included Asian (CHIK/Indonesia/0811aTW/2008/FJ807891), failed to make body titer data normally distributed. The and CESA (CHIK/Singapore/0611aTW/2006/FJ807896, and rank tests (Mann-Whitney U test for two samples or Kruskal- E1/226V CHIK/Malaysia/0901aTW/2009/FJ807895), which Wallis tests for multiple samples) were used to detect the were isolated from blood specimens of the imported Chi- difference in body titers among mosquito species, virus kungunya cases from Indonesia, Singapore, and Malaysia. All strains, and postinfection days.29,30 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 Results was used for the following experiments and this concen- tration was close to the high virus load (ranging from The fitness of CHIKV in laboratory-reared Aedes 4.69 103 PFU/mL to 5.62 108 PFU/mL) of viremic pa- albopictus and Aedes aegypti tients.25 A total of 10e20 female mosquitoes were placed in a small paper cup (8 cm diameter 9.5 cm height; Yeong First of all, the virus titer standard curve was performed by Hung Trading Co., LTD, Taichung, Taiwan) covered with fine real time PCR to estimate the virus titers in the orally- nylon mesh and one drop (1 mL) of the feeding mixture was infected mosquitoes, and the linear regression of Ct value (Y) placed on the mesh of each cup. Mosquitoes were allowed to against log (viral load) (x) was Y Z 4.119 log(X) þ 36.77, feed for 90 minutes with new blood drops being added every with the R square value of 1.00. 30 minutes.26,27 The mosquitoes were then held in a growth We evaluated the viral fitness of the Asian genotype chamber at 28 C and 75% relative humidity (RH). Cotton CHIKV in both the laboratory-reared Ae. albopictus and Ae. soaked in a 10% sugar solution was provided on the mesh. aegypti in infectious blood at a titer of 1.98 106 PFU/mL. Mosquitoes were frozen at 0 day, 1 day, 2 day, 3 day, 4 day, 5 Only body titers of infected Ae. aegypti on 1 day post day, 6 day, 12 day, 18 day, and 24 day intervals until they infection (dpi) (102.20) and 4 (102.76) were significantly died, and then they were stored at 80 C until being pro- (Z Z 3.84, df Z 1, p < 0.001; Z Z 2.25, df Z 1, p < 0.05) cessed for virus detection. 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 Virus detection and titration copy slightly decreased, the mean (standard deviation) of viral genome at Day 0 was 103.08 0.17, but the viral copy A single mosquito was homogenized and clarified by number ranged from 102.12 0.23 to 102.76 0.88 at an early centrifugation (9560g). Viral RNA (70 mL) was extracted infectious stage (1e4 dpi; Fig. 2A). CHIKV genome amplifi- from 140 mL of mosquito suspension using the QIAamp viral cation started increasing and peaked at 5 dpi (103.87 1.1). RNA mini kit (catalog number 52,906, Qiagen, Hilden, Then, it decreased over time at 8 dpi, 10 dpi, and 12 dpi Germany) according to the manufacturer’s instructions. (103.17 1.24,103.14 1.66, and 102.55 1.64, respectively). Amplification by reverse transcription polymerase chain The viral genome bounced back slightly at 18 dpi and 24 dpi reaction (RT-PCR) was performed using the Mx4000 quan- (103.30 1.47 and 103.12 1.43, respectively). The viral titative PCR system (Stratagene, La Jolla, CA, USA). Sam- growth curve in Ae. albopictus shows that the viral genome m ples were assayed in a 50 L reaction mixture containing copy decreased (range from 101.22 1.06 to 102.28 0.48)at m 10 L of sample RNA and optimal concentrations of the the first 4 days after infection (Fig. 2B). CHIKV increased in primers using the QuantiTect SYBR Green RT-PCR kit (cat- mosquitoes at 6 dpi (103.11 1.38) and peaked at 10 dpi alog number 204,243, Qiagen). Alphavirus-specific primers 3.63 1.05 0 0 0 (10 ). After 18 dpi, the viral genome largely (AL-2: 5 -TAA TGC CAG AGC GTT TTC GCA-3 , AL-3: 5 -GTG decreased (103.06 0.86). 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 The growth curves of non-variant CHIKV and variant (nsp1) genes to detect all alphaviruses, and the products E1/226V strains in wild Aedes albopictus were expected to be 414 bp. The thermal profile consisted populations of a 30-minute reverse transcription step at 50 C and 15 minutes of Taq polymerase activation at 95 C, followed by Next, we tested susceptibilities of the nonvariant and E1/ 45 cycles of PCR (94 C for 15 seconds, annealing tempera- 226V variant CESA CHIKV in field-collected Ae. albopictus ture 55 C for 30 seconds, 72 C for 20 seconds, and 77 C for from Northern (Taipei), Central (Taichung), Eastern (Hua- 30 seconds). Following amplification, a melting curve lien), and Southern (Kaohsiung) Taiwan. For the nonvariant analysis was performed to verify the correct product using strain, body titers of four local mosquito strains were its specific melting temperature. Additionally, serial 10-fold significantly (p < 0.05) different at 0 dpi, 1 dpi, 4 dpi, 12 dilutions were undertaken of CHIKV (CESA strain) with an dpi, 18 dpi, and 24 dpi. For the variant strain, body titers initial viral load of w106 PFU/mL (determined by plaque of these four local mosquito strains were significantly assay). Each dilution was added to 140 mL of one nonin- different (p < 0.05) at all dpi. The nonvariant viral copy fected Ae. aegypti female mosquito suspension to estimate numbers were increased at 1 dpi (104.18 0.41,103.68 0.51) the virus titers in the orally-infected mosquitoes. in the Taichung and Hualien strains, respectively (Fig. 3A Susceptibility of A. albopictus to Chikungunya virus 549

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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 Thailand, Myanmar, Singapore, Indonesia, and China during viral copy number increased at 2 dpi (103.87 0.92 and 2008e2013. Although no local CHIKV outbreaks occurred in 103.95 0.65; Fig. 3E and G). Since Day 4, viral genome Taiwan, the introductions of the CHIKV viruses were iden- amplification largely increased, with the viral genome tified from time to time through the imported cases. number of 105.54 1.01,105.67 0.86, and 104.76 0.64 in the Therefore, we infected Ae. albopictus and Ae. aegypti with Taichung, Hualien, and Taipei populations, respectively, three imported CHIKV isolates in the laboratory. The virus and at 5 dpi in Kaohsiung captured mosquito (105.06 0.87). susceptibility varied with virus strain and mosquito species The maximum virus yields for Taichung, Hualien, Taipei, and strain. The Asian virus strain started to replicate at 5e6 and Kaohsiung mosquitoes were 105.67 0.86 at 6 dpi, dpi with the maximum virus yield, ranging from 103.63 to 106.27 0.40 at 12 dpi, 105.66 0.23 at 12 dpi, and 105.51 0.39 103.87 at 5e10 dpi in both species. The variant CHIKV CESA at 12 dpi, respectively. virus genotype replicated earlier at 1 dpi with the maximum After mosquitoes were infected with the variant virus virus yield ranging from 105.63 to 106.52 at 3e6 dpi in Ae. strain, the virus replicated immediately, and all the viral albopictus females while the nonvariant virus strain repli- copy numbers were higher than that at 0 dpi in all Ae. cated at 1e2 dpi with the maximum virus yield ranging from albopictus mosquitoes at 1 dpi (viral RNA ranging from 105.51 to106.27 at 6e12 dpi. In Ae. aegypti, these viruses 103.93 0.57 to 104.71 0.46; Fig. 3B, D, F, and H). The viral replicated at 1e2 dpi with the maximum yields at 4e5 dpi growth curve reached its peak at 6 dpi in Taichung (range from 105.38 to 105.62). Therefore, we concluded that (106.09 0.35), 5 dpi in Hualien (106.23 0.52), and 4 dpi in the risk of CHIKV in Taiwan is high in all distribution areas of Taipei (105.73 0.27) captured mosquitoes but peaked at 3 Ae. aegypti and Ae. albopictus and that the variant virus dpi (105.38 0.43) in Kaohsiung mosquitoes. strain presents an even higher risk. We also evaluated susceptibilities of the nonvariant and In our study, susceptibility of the CHIKV varied by virus E1/226V variant CHIKV in the laboratory-reared Ae. aegypti strain and mosquito species and strain, similar to studies of to provide a comparison with the results of Ae. albopictus. the CHIKV from American countries30 and the dengue Body titers of the nonvariant and E1/226V variant were not virus.31 Some investigations have indicated that Ae. albo- significantly different (p > 0.10) on all dpi for Ae. aegypti. pictus is more susceptible than Ae. aegypti to the variant However, body titers of infected Ae. aegypti were signifi- type CHIKV,32 which is similar to three mosquito strains in cantly lower (p < 0.05) than those of some Ae. albopictus our results. In the Kaohsiung mosquito strain, virus repli- strains (Taichung and Hualien strains) for some dpi. The cation did not consistently occur. Previous studies have also viral growth curve of nonvariant CHIKV decreased at 1 dpi shown that the variant strain CHIKV produced high viremia (103.32 0.53) compared to 0 dpi (103.46 0.26; Fig. 4A). It in monkeys, sufficient to infect Ae. albopictus and Ae. started increasing at 2 dpi (104.25 1.39) and peaked at 4 dpi aegypti.33 In this study, E1/226V variant and nonvariant (105.38 1.04). Then, the number of viral RNA stayed steady CHIKV infection rates were consistently higher in all strains until Day 24 pi. Additionally, the viral growth kinetics of the of Ae. albopictus tested than in all strains of Ae. aegypti variant CHIKV indicated that the viral genome copy slightly tested (Figs. 3 and 4). Additionally, the E1/226V CHIKV increased (from 103.48 0.18 to 103.87 1.17) at the early replicated at 1e2 dpi earlier than the nonvariant type virus infectious stage (0e2 dpi; Fig. 4B). It largely replicated at 3 and the Asian genotypes, indicating that the extrinsic in- dpi (105.08 0.63) and peaked at 5 dpi (105.62 0.64). cubation period may be shorter and indicating efficient transmission to other hosts. Determination of vector competence of mosquito pop- Discussion ulations is a key parameter in evaluating the risk of CHIKV transmission and spread. Vector competence is determined The E1/226V variant strains of CESA genotype have spread by the preference of mosquito species on human blood and from Africa and India to other parts of the world and caused virus replication in mosquito vectors including viral load and epidemics in Southeast Asian countries including Malaysia, speed of replication. Both Ae. aegypt and Ae. albopictus 550 T.-H. Chen et al.

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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. Susceptibility of A. albopictus to Chikungunya virus 551

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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 containers infested with Aedes immatures per 100 houses), speed of replication represents the duration of the extrinsic and container index (the percentage of water-holding incubation period. No papers were available on the corre- containers infested with Aedes immatures). The dengue lation of the viral load in mosquitoes to the actual occur- control strategies include source reduction and insecticide rence of transmission and/or outbreaks in vivo. However, sprays, in which the application frequencies of insecticides high viral loads of CHIKV were commonly detected both in were based on extrinsic incubation period in mosquitoes.40 the field-collected mosquito samples (3.26 104 copies/ Therefore, the rapid replication of CHKV in our study will mL) and human patient samples on Day 1 of fever onset affect the frequencies of insecticide sprays from a week in (8.57 106 copies/mL) during outbreaks, such as a Chi- dengue control to 4 days to battle the Chikungunya. kungunya 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. Acknowledgments In Taiwan, the dengue virus is introduced from abroad by infected travelers during the early summer each year. This study was supported by scientific research grants from Later, the virus is vectored locally by mosquitoes and usu- the Centers for Disease Control, Taiwan in 2009e2011 ally disappears during the dry and cold winter season. The (DOH98-DC-2013, DOH99-DC-2030, and DOH100-DC-2018). 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 References cases of Chikungunya in Taiwan have been reported, the possibility cannot be excluded due to the similar symptoms 1. Lumsden WH. An epidemic of virus disease in Southern Prov- 37 of these two diseases. Historical reports indicate that ince, Tanganyika Territory, in 1952e53. II. General description some epidemics caused by the dengue virus may also have and epidemiology. Trans R Soc Trop Med Hyg 1955;49:33e57. been caused by CHIKV.38,39 In Taiwan, the CHIKV virus 2. Powers AM, Logue CH. Changing patterns of Chikungunya virus: strains isolated from imported cases include the Asian, E1- re-emergence of a zoonotic arbovirus. J Gen Virol 2007;88: 226V variant, and nonvariant CESA genotypes.21 Therefore, 2363e77. it is very likely that this pattern of dengue transmission 3. Robinson MC. An epidemic of virus disease in Southern Prov- ince, Tanganyika Territory, in 1952e53. I. Clinical features. could also occur with CHIKV. Mosquito control is the sole Trans R Soc Trop Med Hyg 1955;49:28e32. available method for reducing transmission of Chikungunya, 4. Brighton SW, Prozesky OW, de la Harpe AL. Chikungunya virus and no vaccines are available. Control of this disease, as infection. A retrospective study of 107 cases. S Afr Med J 1983; with dengue, is highly dependent on source reduction to 63:313e5. reduce vector density to prevent the occurrence of this 5. Jaffar-Bandjee MC, Ramful D, Gauzere BA, Hoarau JJ, Krej- disease.1,2 Fortunately, this control method is working for bich-Trotot P, Robin S, et al. Emergence and clinical insights CHIKV as well as for dengue.35,36 Finally, we conclude that into the pathology of Chikungunya virus infection. Expert Rev the risk of CHIKV in Taiwan is high in all distribution areas of Anti Infect Ther 2010;8:987e96. Ae. aegypti and Ae. albopictus for the CESA genotype, and 6. Dupuis-Maguiraga L, Noret M, Brun S, Le Grand R, Gras G, that the variant virus strain presents an even higher risk. Roques P. Chikungunya disease: infection-associated markers from the acute to the chronic phase of arbovirus-induced Dengue vector surveillance in Taiwan was conducted by arthralgia. PLoS Negl Trop Dis 2012;6:e1446. local health bureaus and population density is represented 7. Strauss JH, Strauss EG. The alphaviruses: gene expression, as adult index (the number of Aedes adults, Ae. aegypti or replication, and evolution. Microbiol Rev 1994;58:491e562. Ae. albopictus per 100 houses) or traditional Stegomyia 8. Yergolkar PN, Tandale BV, Arankalle VA, Sathe PS, Sudeep AB, indices, house index (the percentage of houses infested Gandhe SS, et al. Chikungunya outbreaks caused by African with Aedes immatures), Breteaux index (the number of genotype, India. Emerg Infect Dis 2006;12:1580e3. 552 T.-H. Chen et al.

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