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Surveillance of Encephalitis-Causing Arboviruses in Horses in South Korea
Journal of Equine Veterinary Science 37 (2016) 11–16
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Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Surveillance of Encephalitis-Causing Arboviruses in Horses in South Korea Hye-Young Jeoung a, Sun-Ju Yang b, Yun-ki Choi b, Ji-Hye Lee a, Hyun-Ji Seo a, Sung-Hee Kim a, Yun Sang Cho a, Yong-Joo Kim a, In-Soo Cho a, Jee-Yong Park a, * a b
Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do, Republic of Korea Equine Hospital, Korea Racing Authority, Gwacheon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2015 Received in revised form 14 October 2015 Accepted 2 November 2015 Available online 11 November 2015
Surveillance was conducted in South Korea to look for evidence of infection with Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV) in horses from 2012 to 2013. The surveillance consisted of passive surveillance of testing horses with neurologic symptoms such as paralysis, incoordination, ataxia and circling, and active surveillance of testing for serologic evidence of infection in healthy horses. Passive surveillance was conducted for EEEV, WEEV, VEEV, WNV, SLEV, and JEV, and whole blood and/or brain samples received from 49 horses with neurologic signs were tested by polymerase chain reaction (PCR). Active surveillance was conducted for WNV and JEV, and 2,695 serum samples collected from horses across the country were tested for antibodies by IgM enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT), respectively. All samples tested by PCR were negative for EEEV, WEEV, VEEV, WNV, and SLEV, except for one whole blood sample (1/45) that was positive for JEV. All samples tested for WNV antibodies were shown to be negative. For JEV, because South Korea is endemic and horses in South Korea are vaccinated against JEV, various titers of antibodies for JEV were detected by VNT in 57.8% (737/1,274) and 58.9% (837/1,421) of the sera in 2012 and 2013, respectively. The surveillance provides evidence that supports the view that South Korea is free from EEEV, WEEV, VEEV, WNV, and SLEV. In addition, the surveillance scheme was shown to be able to identify JEV infections in the equine population and provide serologic data for JEV that could be used to improve the current vaccination program for JEV in horses. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Surveillance Arbovirus Zoonosis Encephalitis Horse
1. Introduction Equine encephalitis is characterized by signs of central nervous system dysfunction with moderate to high mortality [1], and arboviruses belonging to Togaviridae (genus Alphavirus) and Flaviviridae (genus Flavivirus) are the most common cause of equine viral encephalitis [2]. A number of * Corresponding author at: Jee-Yong Park, Animal and Plant Quarantine Agency, 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-824, Republic of Korea. E-mail address: [email protected] (J.-Y. Park). 0737-0806/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.11.004
alphaviruses including Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV) are distributed across the United States, whereas flaviviruses such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV) are reported in many parts of the world except for SLEV, which has only been reported in the United States [2–4]. South Korea is considered to be free from infection with EEEV, WEEV, VEEV, WNV, and SLEV, although antibodies against WNV have been detected in migratory waterfowl [5]. Japanese encephalitis virus is endemic in South Korea and has been
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H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16
detected in domestic animals such as horses, wild birds, mosquitoes as well as humans [6–9]. Many of these viruses such as EEEV, WEEV, VEEV, WNV, SLEV, and JEV are zoonotic and have the potential to emerge or reemerge in many parts of the world due to increased trade, increased movement of people and animals, and climate change [10– 15]. Therefore, surveillance of arbovirus-associated encephalomyelitis was conducted in horses, which consisted of passive surveillance for EEEV, WEEV, VEEV, WNV, SLEV, and JEV by testing blood and/or brain tissue samples from horses with neurologic signs by antigen tests and active surveillance for WNV and JEV by testing sera with antibody tests. The surveillance was conducted to look for evidence of infection with arboviruses and to establish a test strategy that can quickly detect new introductions and elucidate the situation of equine encephalitis in South Korea. 2. Materials and Methods 2.1. Sample Collection Passive surveillance was conducted for EEEV, WEEV, VEEV, WNV, JEV, and SLEV in clinically suspect cases of horses with neurologic signs, such as paralysis, incoordination, ataxia, and circling. Brain and/or whole blood samples from suspect cases were collected and submitted by field veterinarians to the Animal and plant Quarantine Agency for antigen testing. Samples were received between May and October of 2012 and 2013, which were from a total of 49 horses from Gyeongnam (12 affected horses from 11 farms), Busan (17 affected horses from 10 farms), Gyeongbuk (8 affected horses from 8 farms), Gyeonggi (9 affected horses from 7 farms), Jeonnam (2 affected horses from 2 farms), and Ulsan (1 affected horse from 1 farm). Brain samples consisting of forebrain, midbrain, hindbrain, cerebellum, brainstem, and spinal cord were collected from five horses during necropsy, and whole blood samples were received from 45 horses (Table 1) in plastic tubes with spray-coated K2EDTA (BD, cat, 367488). Horses with samples positive by reverse transcription–polymerase chain reaction (RT-PCR) were considered to be positive cases. Active surveillance was conducted for WNV and JEV to look for evidence of infection in sera collected from clinically normal horses by antibody detection tests. Serum samples were collected through farm visits by the staff of the Korean Racing Authority, a public corporation under the Ministry of Agriculture, Food and Rural Affairs. A total of 2,695 serum samples were collected throughout South Korea (Fig. 1) between May and October of 2012 and 2013 (Table 2). 2.2. RNA Extraction and Identification of the Agents Total viral RNA was extracted from samples using Maxwell 16 research instrument system (Promega, Medison, WI) with Maxwell 16 LEV simply RNA Tissue Kit (Promega, cat, AS1280) and Maxwell 16 LEV simply RNA Blood Kit (Promega, cat, AS1310), according to the manufacturer’s instructions. Extracted RNA was tested using RT-PCR for EEEV, WEEV [16], and VEEV [17], and by multiplex real-time RT-PCR for WNV, JEV, and SLEV [18]. For positive controls,
Korean JEV Anyang 300 strain, and WNV NY 385-99 strain (ATCC VR-1507), which was obtained from the American Type Culture Collection (ATCC, Manassas, VA), were used. For the rest of the viruses, the respective target genes were synthesized (Bioneer, South Korea) and used as controls. 2.3. Antibody Test for WNV The serum samples were tested for WNV antibodies using ID Screen West Nile IgM antibody capture ELISA (ID VET, France). Samples were considered positive if the S/P % was 45%, according to the manufacturer’s instructions. 2.4. Antibody Test for JEV Serum samples were inactivated at 56 C for 30 minutes before analysis. Neutralizing JEV antibodies were detected in collected sera using an in vitro neutralization assay as described previously [19,20]. For in vitro neutralization assay, JEV (Anyang 300 strain) was maintained in Vero cells. All cell lines were grown in Dulbecco’s modified Eagle’s medium (GibcoBRL, Gaithersburg, MD) supplemented with 5% heat-inactivated fetal bovine serum (GibcoBRL) in a humidified 5% CO2 atmosphere at 37 C. For this study, different criteria were used for the determination of JEV infection for vaccinated and nonvaccinated horses. For horses with record of JEV vaccination, JEV infection was considered if VN titers were equal or above 1:640. This was based on previous reports that showed horses inoculated with inactivated JEV vaccines demonstrated antibody titers of less than 1: 640, which then increased to above 1:640 when the horses were experimentally inoculated with JEV 2 to 3 weeks after vaccination [19]. Japanese encephalitis virus–vaccinated horses with VN titers between 1:10 and 1:640 were considered to be due to vaccination and negative for antibodies to JEV if sera exhibited titers below 1:10. For horses that did not have record of vaccination, JEV infection was considered when titers were equal or above 1:160, based on previous report of horses with JEV infection demonstrating VN antibody titer from 1:160 to 1:640 [20]. NonJEV–vaccinated horses with VN titers between 1:10 and 1:80 were considered suspect, and those with below 1:10 were considered negative. 3. Results 3.1. Passive Surveillance Samples collected from horses with clinical signs indicative of arbovirus-associated encephalitis such as fever, depression, loss of appetite, paralysis, ataxia, circling, incoordination, and vision disorder [1,3,21] were tested. All samples collected from the 49 horses tested negative for EEEV, WEEV, VEEV, WNV, and SLEV by PCR. Japanese encephalitis virus was detected in one equine blood sample (1/45, sample number 13-2) by multiplex real-time RT-PCR. The positive sample was from a 5 -year-old mare located in Gyeongnam that had neurologic symptoms including hind paralysis. Japanese encephalitis virus was confirmed by sequencing PCR products by which the correct targets were amplified [22].
H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16
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Table 1 The region, number of affected horses, neurologic signs, and samples submitted by premises. Premises
Region
No. Horses
Neurologic Signs
Samples
12-01 12-02 12-03 12-04 12-05 12-06 12-07 12-08 12-09 12-10 12-11 12-12 12-13 12-14 12-15 12-16 12-17 12-18 12-19 12-20 12-21 12-22 12-23 12-24 12-25 12-26 12-27 12-28 12-29 12-30 12-31 13-01 13-02 13-03 13-04 13-05 13-06 13-07 13-08
Gyeongbuk Busan Busan Gyeonggi Busan Gyeongbuk Busan Busan Gyeonggi Gyeonggi Gyeongbuk Ulsan Gyeongnam Gyeonggi Gyeonggi Busan Busan Gyeongnam Gyeonggi Jeonnam Gyeongnam Gyeongnam Gyeongnam Gyeongbuk Gyeongbuk Gyeongbuk Busan Gyeongnam Gyeongnam Gyeongnam Gyeongnam Gyeongbuk Gyeongnam Gyeongnam Busan Gyeongbuk Busan Jeonnam Gyeonggi
1 1 1 3 1 1 5 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1
Front and hind ataxia Hind ataxia Hind paralysis Hind paralysis, convulsion Hind ataxia Fore and hind limb ataxia Depression, nasal discharge, fever, cough Hind ataxia Hind ataxia Hind ataxia Hind paralysis Hind ataxia Hind ataxia, weight loss Neurologic disorder Ataxia Incoordination, circling Incoordination, circling Hind paralysis Ataxia, anorexia, blindness, loss of appetite Hind ataxia Hind paralysis Hind paralysis Hind paralysis Hind paralysis Hind paralysis Hind paralysis, death Hind paralysis Incoordination Hind paralysis Anorexia, fever Hind paralysis Incoordination Hind paralysis Incoordination Fever Hind paralysis Hind paralysis Hind paralysis Hind paralysis
Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Brain Whole Whole Brain Brain Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Brain Whole
blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood Brain blood blood
blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood
Abbreviations: EEEV, Eastern equine encephalitis virus; JEV, Japanese encephalitis virus; SLEV, St. Louis encephalitis virus; VEEV, Venezuelan equine encephalitis virus; WEEV, Western equine encephalitis virus. For passive surveillance, clinically suspect cases of horses with neurologic signs were submitted for etiologic findings to the Animal and plant Quarantine Agency from 2012 to 2013 and tested for EEEV, WEEV, VEEV, SLEV, and JEV.
3.2. Active Surveillance Serologic testing showed all serum samples tested for WNV antibodies were negative by ID Screen West Nile IgM antibody capture ELISA (ID VET, France). From the 1,274 sera collected in 2012, a total of 1,165 samples (91.4%) were from horses with record of JEV vaccination, of which 8 samples (0.7%) had VNT titers equal or above 1:640, which indicates JEV infection as designated by the criteria set for this study. In addition, of the 1,165 samples from horses that were vaccinated, 497 samples (42.7%) showed VNT titers below 1:10, indicating these horses were negative for antibodies to JEV. A total of 109 samples were from horses which were recorded as nonvaccinated for JEV, of which 5 (4.6%) were equal or above 1:160 (Table 3). For the 1,421 sera collected in 2013, a total of 1,127 samples (79.3%) were from horses with record of JEV vaccination, of which 13 samples (1.2%) had VNT titers equal or above 1:640, and 455 samples (40.4%) were below 1:10. A total of 294 samples were from horses with no
record of JEV vaccination, of which 36 (12.2%) were equal or above 1:160. 4. Discussion Global warming, growing urbanization, faster transportation, increased animal movements, and rapid spread of arthropod vectors all contribute to the continuous spreading of arboviruses into new geographic areas [3,15]. Many arboviruses were originally found in tropical regions such as Africa, South America, and in parts of Asia, but the geographic distribution and frequency of epidemic outbreaks of arboviral diseases have expanded dramatically across the world in the past several decades [23]. For example, in the case of EEE, which has been reported along the eastern coast of the United States and Canada, there have been an increase in the number of human EEE infections in the northernmost part of the outbreak areas [24]. Western equine encephalitis virus and VEEV have also been reported throughout most parts of the United States
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H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16 Table 2 Serologic test results for antibodies to Japanese encephalitis virus in horse sera.
Gyeonggi
Regions
Gangwon Seoul Incheon
Number of JEV-Positive Serum/Total Number of Serum (%) 2012
Chungbuk Chungnam Daejon
Jeonbuk
Gyeongbuk
Daegu Ulsan
Gyeongnam Gwangju
Busan
Jeonnam
Seoul Incheon Busan Daejeon Gwangju Daegu Ulsan Gyeonggi Gangwon Chungbuk Chungnam Jeonbuk Jeonnam Gyeongbuk Gyeongnam Jeju Total
164/277 0/2 165/199 3/3 6/7 5/5 d 135/363 6/8 11/12 10/11 61/91 9/13 7/11 1/1 154/271 737/1,274
2013 (59.2) (0) (82.9) (100) (85.7) (100) (37.1) (75) (91.7) (90.9) (67) (69.2) (63.6) (100) (56.8) (57.8)
d d 148/175 d d d 5/5 159/378 d d d 81/109 d 133/144 12/14 299/596 837/1,421
(84.6)
(100) (42.1)
(74.3) (92.4) (85.7) (50.2) (58.9)
Abbreviations: JEV, Japanese encephalitis virus; WNV, West Nile virus. For active surveillance, a total of 2,695 serum samples from clinically normal horses were collected from 2012 to 2013 throughout South Korea and serologically tested for WNV and JEV antibodies.
Jeju Fig. 1. Geographical regions of South Korea.
including Brazil and Argentina [25–28]. West Nile virus was first isolated in Africa but is now reported worldwide including the Middle East, Asia, Australia, and the United States [3]. Japanese encephalitis virus, which had been confined to countries in Asia, has since been reported in Australia [29]. For an arbovirus transmission cycle to become established, three factors are essential, which are the arbovirus, arthropod, and the vertebrate host, and if all factors are available in sufficient numbers, the virus will continuously circulate between the arthropod vector and competent host [30,31]. In South Korea, several species of mosquitoes including Culex spp. and Aedes spp. with the ability to transmit these arboviruses have been identified [6,32]. In addition, the possibility of new introduction of arboviruses such as EEEV, WEEV, VEEV, WNV, and SLEV into South Korea continues to grow due to climate change, increase in international movement of commodities and animal movements, and migratory birds [5]. Should these arboviruses become introduced into South Korea, it is possible that they could become quickly established due to the presence of viable vectors and hosts. In addition, because large proportion of the hosts would be naive to the viruses, huge economic losses could be expected due to large number of clinical cases. Therefore, it is important that South Korea conducts surveillance for arboviruses, focusing on viruses belonging to the families Togaviridae (genus Alphavirus) and Flaviviridae (genus Flavivirus), as these viruses are zoonotic and are associated with encephalitis, often with high mortality in humans as well as horses [2,33]. For these reasons, the present study
consisting of passive and active surveillance was conducted to determine the situation of major encephalitis-causing arboviruses in South Korean horses. No evidence of EEEV, WEEV, VEEV, WNV, and SLEV was found by passive surveillance, which supported the view that South Korea is free from these diseases. South Korea is endemic for JEV, and through passive surveillance conducted in this study, a JEV-positive horse was diagnosed. In addition, an active surveillance of healthy horses was conducted as part of the study, and 2,695 horses from across the country were serologically tested for WNV and JEV antibodies. For WNV serosurveillance, WNV ID Screen West Nile IgM antibody capture ELISA (ID VET, France) was used, which has a narrower window of detection compared with tests that detect IgG antibodies. However, for South Korea, which is not only endemic for JEV but also vaccinates horses for JEV, tests that detect IgG antibodies such as IgG ELISA were inappropriate due to cross-reactivity with JEV antibodies. Because horses in South Korea are regularly vaccinated to prevent infection of JEV [8,9,34], for the purpose of this study, a criteria was established to determine the JEV infection status based on the VN titers for vaccinated and nonvaccinated horses in South Korea. A total of 2,292 horses (85%) had a record of JEV vaccination, and 21 (0.9%) of these horses were positive for JEV infection, suggesting that they were infected with JEV but have since recovered or the infection was subclinical, perhaps due to protective immunity conferred by vaccination. Interestingly, of the vaccinated horses, 952 (41.5%) were JEV antibody negative, suggesting that a large percentage of the vaccinated horses had not seroconverted and that the current vaccination strategy for JEV may need to be reviewed. A total of 403 samples were from horses which were recorded as nonvaccinated for JEV, and 41 (10.2%) of these horses that had VN titers equal or above 1:160 were likely to have been infected with JEV, whereas 193 horses (47.9%) with VN titers between 1:10 and 1:80,
H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16
15
Table 3 Antibody titers against JEV by virus neutralization test in South Korean horses. Year
JEV Vaccination
Total No. of Samples
JE Antibody Titers <10
10
20
40
80
160
320
640
>640
2012
Vaccinated Nonvaccinated Total Vaccinated Nonvaccinated Total
1,165 109 1,274 1,127 294 1,421
497 40 537 455 129 584
148 14 162 124 26 150
164 18 182 135 36 171
153 18 171 150 27 177
110 14 124 117 40 157
66 2 68 82 21 103
19 2 21 51 12 63
7 1 8 10 3 13
1 0 1 3 0 3
2013
Abbreviation: JEV, Japanese encephalitis virus.
which were designated as being suspect could be considered to be due to infection with JEV but may also be due to nonspecific reaction to unknown factors. Another confounding factor that could not be overlooked was the possibility that some of the horses recorded as nonvaccinated had in fact been vaccinated but was not recorded as such, which would account for the presence of JEV antibodies in these horses. Future surveillance with a more strict recording system will be needed to better understand the JEV situation in South Korea. 5. Conclusions In this study, a surveillance of arbovirus-associated equine encephalitis was conducted, which could provide supporting evidence to demonstrate freedom from major arboviral diseases and was able to detect encephalitiscausing arboviral infection in horses such as JEV infection. Serologic surveillance demonstrated the possible presence of subclinical infection of JEV in the equine population and identified the need to review the current JEV vaccination strategy. Continued surveillance will be required to maintain the ability to quickly detect any new introductions and to provide information on the situation of equine encephalitis in South Korea. Acknowledgments This work was supported by a grant (Project Code No. P1541782-2012-12-02) from the Animal and Plant Quarantine Agency and Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and the authors are grateful to the National park research institute migratory bird center for their support in collection of dead wild birds. References [1] Keane DP, Little PB. Equine viral encephalomyelitis in Canada: a review of known and potential causes. Can Vet J 1987;28:497–504. [2] Davis LE, Beckham JD, Tyler KL. North American encephalitic arboviruses. Neurol Clin 2008;26:727–57. ix. [3] Go YY, Balasuriya UB, Lee CK. Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses. Clin Exp Vaccine Res 2014;3:58–77. [4] Spinsanti LI, Diaz LA, Glatstein N, Arselan S, Morales MA, Farias AA. Human outbreak of St. Louis encephalitis detected in Argentina, 2005. J Clin Virol 2008;42:27–33. [5] Yeh JY, Park JY, Ostlund EN. Serologic evidence of West Nile Virus in wild ducks captured in major inland resting sites for migratory waterfowl in South Korea. Vet Microbiol 2011;154:96–103.
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Short Communication
Surveillance of Encephalitis-Causing Arboviruses in Horses in South Korea Hye-Young Jeoung a, Sun-Ju Yang b, Yun-ki Choi b, Ji-Hye Lee a, Hyun-Ji Seo a, Sung-Hee Kim a, Yun Sang Cho a, Yong-Joo Kim a, In-Soo Cho a, Jee-Yong Park a, * a b
Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do, Republic of Korea Equine Hospital, Korea Racing Authority, Gwacheon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2015 Received in revised form 14 October 2015 Accepted 2 November 2015 Available online 11 November 2015
Surveillance was conducted in South Korea to look for evidence of infection with Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV) in horses from 2012 to 2013. The surveillance consisted of passive surveillance of testing horses with neurologic symptoms such as paralysis, incoordination, ataxia and circling, and active surveillance of testing for serologic evidence of infection in healthy horses. Passive surveillance was conducted for EEEV, WEEV, VEEV, WNV, SLEV, and JEV, and whole blood and/or brain samples received from 49 horses with neurologic signs were tested by polymerase chain reaction (PCR). Active surveillance was conducted for WNV and JEV, and 2,695 serum samples collected from horses across the country were tested for antibodies by IgM enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT), respectively. All samples tested by PCR were negative for EEEV, WEEV, VEEV, WNV, and SLEV, except for one whole blood sample (1/45) that was positive for JEV. All samples tested for WNV antibodies were shown to be negative. For JEV, because South Korea is endemic and horses in South Korea are vaccinated against JEV, various titers of antibodies for JEV were detected by VNT in 57.8% (737/1,274) and 58.9% (837/1,421) of the sera in 2012 and 2013, respectively. The surveillance provides evidence that supports the view that South Korea is free from EEEV, WEEV, VEEV, WNV, and SLEV. In addition, the surveillance scheme was shown to be able to identify JEV infections in the equine population and provide serologic data for JEV that could be used to improve the current vaccination program for JEV in horses. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Surveillance Arbovirus Zoonosis Encephalitis Horse
1. Introduction Equine encephalitis is characterized by signs of central nervous system dysfunction with moderate to high mortality [1], and arboviruses belonging to Togaviridae (genus Alphavirus) and Flaviviridae (genus Flavivirus) are the most common cause of equine viral encephalitis [2]. A number of * Corresponding author at: Jee-Yong Park, Animal and Plant Quarantine Agency, 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-824, Republic of Korea. E-mail address: [email protected] (J.-Y. Park). 0737-0806/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.11.004
alphaviruses including Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV) are distributed across the United States, whereas flaviviruses such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV) are reported in many parts of the world except for SLEV, which has only been reported in the United States [2–4]. South Korea is considered to be free from infection with EEEV, WEEV, VEEV, WNV, and SLEV, although antibodies against WNV have been detected in migratory waterfowl [5]. Japanese encephalitis virus is endemic in South Korea and has been
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detected in domestic animals such as horses, wild birds, mosquitoes as well as humans [6–9]. Many of these viruses such as EEEV, WEEV, VEEV, WNV, SLEV, and JEV are zoonotic and have the potential to emerge or reemerge in many parts of the world due to increased trade, increased movement of people and animals, and climate change [10– 15]. Therefore, surveillance of arbovirus-associated encephalomyelitis was conducted in horses, which consisted of passive surveillance for EEEV, WEEV, VEEV, WNV, SLEV, and JEV by testing blood and/or brain tissue samples from horses with neurologic signs by antigen tests and active surveillance for WNV and JEV by testing sera with antibody tests. The surveillance was conducted to look for evidence of infection with arboviruses and to establish a test strategy that can quickly detect new introductions and elucidate the situation of equine encephalitis in South Korea. 2. Materials and Methods 2.1. Sample Collection Passive surveillance was conducted for EEEV, WEEV, VEEV, WNV, JEV, and SLEV in clinically suspect cases of horses with neurologic signs, such as paralysis, incoordination, ataxia, and circling. Brain and/or whole blood samples from suspect cases were collected and submitted by field veterinarians to the Animal and plant Quarantine Agency for antigen testing. Samples were received between May and October of 2012 and 2013, which were from a total of 49 horses from Gyeongnam (12 affected horses from 11 farms), Busan (17 affected horses from 10 farms), Gyeongbuk (8 affected horses from 8 farms), Gyeonggi (9 affected horses from 7 farms), Jeonnam (2 affected horses from 2 farms), and Ulsan (1 affected horse from 1 farm). Brain samples consisting of forebrain, midbrain, hindbrain, cerebellum, brainstem, and spinal cord were collected from five horses during necropsy, and whole blood samples were received from 45 horses (Table 1) in plastic tubes with spray-coated K2EDTA (BD, cat, 367488). Horses with samples positive by reverse transcription–polymerase chain reaction (RT-PCR) were considered to be positive cases. Active surveillance was conducted for WNV and JEV to look for evidence of infection in sera collected from clinically normal horses by antibody detection tests. Serum samples were collected through farm visits by the staff of the Korean Racing Authority, a public corporation under the Ministry of Agriculture, Food and Rural Affairs. A total of 2,695 serum samples were collected throughout South Korea (Fig. 1) between May and October of 2012 and 2013 (Table 2). 2.2. RNA Extraction and Identification of the Agents Total viral RNA was extracted from samples using Maxwell 16 research instrument system (Promega, Medison, WI) with Maxwell 16 LEV simply RNA Tissue Kit (Promega, cat, AS1280) and Maxwell 16 LEV simply RNA Blood Kit (Promega, cat, AS1310), according to the manufacturer’s instructions. Extracted RNA was tested using RT-PCR for EEEV, WEEV [16], and VEEV [17], and by multiplex real-time RT-PCR for WNV, JEV, and SLEV [18]. For positive controls,
Korean JEV Anyang 300 strain, and WNV NY 385-99 strain (ATCC VR-1507), which was obtained from the American Type Culture Collection (ATCC, Manassas, VA), were used. For the rest of the viruses, the respective target genes were synthesized (Bioneer, South Korea) and used as controls. 2.3. Antibody Test for WNV The serum samples were tested for WNV antibodies using ID Screen West Nile IgM antibody capture ELISA (ID VET, France). Samples were considered positive if the S/P % was 45%, according to the manufacturer’s instructions. 2.4. Antibody Test for JEV Serum samples were inactivated at 56 C for 30 minutes before analysis. Neutralizing JEV antibodies were detected in collected sera using an in vitro neutralization assay as described previously [19,20]. For in vitro neutralization assay, JEV (Anyang 300 strain) was maintained in Vero cells. All cell lines were grown in Dulbecco’s modified Eagle’s medium (GibcoBRL, Gaithersburg, MD) supplemented with 5% heat-inactivated fetal bovine serum (GibcoBRL) in a humidified 5% CO2 atmosphere at 37 C. For this study, different criteria were used for the determination of JEV infection for vaccinated and nonvaccinated horses. For horses with record of JEV vaccination, JEV infection was considered if VN titers were equal or above 1:640. This was based on previous reports that showed horses inoculated with inactivated JEV vaccines demonstrated antibody titers of less than 1: 640, which then increased to above 1:640 when the horses were experimentally inoculated with JEV 2 to 3 weeks after vaccination [19]. Japanese encephalitis virus–vaccinated horses with VN titers between 1:10 and 1:640 were considered to be due to vaccination and negative for antibodies to JEV if sera exhibited titers below 1:10. For horses that did not have record of vaccination, JEV infection was considered when titers were equal or above 1:160, based on previous report of horses with JEV infection demonstrating VN antibody titer from 1:160 to 1:640 [20]. NonJEV–vaccinated horses with VN titers between 1:10 and 1:80 were considered suspect, and those with below 1:10 were considered negative. 3. Results 3.1. Passive Surveillance Samples collected from horses with clinical signs indicative of arbovirus-associated encephalitis such as fever, depression, loss of appetite, paralysis, ataxia, circling, incoordination, and vision disorder [1,3,21] were tested. All samples collected from the 49 horses tested negative for EEEV, WEEV, VEEV, WNV, and SLEV by PCR. Japanese encephalitis virus was detected in one equine blood sample (1/45, sample number 13-2) by multiplex real-time RT-PCR. The positive sample was from a 5 -year-old mare located in Gyeongnam that had neurologic symptoms including hind paralysis. Japanese encephalitis virus was confirmed by sequencing PCR products by which the correct targets were amplified [22].
H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16
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Table 1 The region, number of affected horses, neurologic signs, and samples submitted by premises. Premises
Region
No. Horses
Neurologic Signs
Samples
12-01 12-02 12-03 12-04 12-05 12-06 12-07 12-08 12-09 12-10 12-11 12-12 12-13 12-14 12-15 12-16 12-17 12-18 12-19 12-20 12-21 12-22 12-23 12-24 12-25 12-26 12-27 12-28 12-29 12-30 12-31 13-01 13-02 13-03 13-04 13-05 13-06 13-07 13-08
Gyeongbuk Busan Busan Gyeonggi Busan Gyeongbuk Busan Busan Gyeonggi Gyeonggi Gyeongbuk Ulsan Gyeongnam Gyeonggi Gyeonggi Busan Busan Gyeongnam Gyeonggi Jeonnam Gyeongnam Gyeongnam Gyeongnam Gyeongbuk Gyeongbuk Gyeongbuk Busan Gyeongnam Gyeongnam Gyeongnam Gyeongnam Gyeongbuk Gyeongnam Gyeongnam Busan Gyeongbuk Busan Jeonnam Gyeonggi
1 1 1 3 1 1 5 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1
Front and hind ataxia Hind ataxia Hind paralysis Hind paralysis, convulsion Hind ataxia Fore and hind limb ataxia Depression, nasal discharge, fever, cough Hind ataxia Hind ataxia Hind ataxia Hind paralysis Hind ataxia Hind ataxia, weight loss Neurologic disorder Ataxia Incoordination, circling Incoordination, circling Hind paralysis Ataxia, anorexia, blindness, loss of appetite Hind ataxia Hind paralysis Hind paralysis Hind paralysis Hind paralysis Hind paralysis Hind paralysis, death Hind paralysis Incoordination Hind paralysis Anorexia, fever Hind paralysis Incoordination Hind paralysis Incoordination Fever Hind paralysis Hind paralysis Hind paralysis Hind paralysis
Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Brain Whole Whole Brain Brain Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Whole Brain Whole
blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood Brain blood blood
blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood
Abbreviations: EEEV, Eastern equine encephalitis virus; JEV, Japanese encephalitis virus; SLEV, St. Louis encephalitis virus; VEEV, Venezuelan equine encephalitis virus; WEEV, Western equine encephalitis virus. For passive surveillance, clinically suspect cases of horses with neurologic signs were submitted for etiologic findings to the Animal and plant Quarantine Agency from 2012 to 2013 and tested for EEEV, WEEV, VEEV, SLEV, and JEV.
3.2. Active Surveillance Serologic testing showed all serum samples tested for WNV antibodies were negative by ID Screen West Nile IgM antibody capture ELISA (ID VET, France). From the 1,274 sera collected in 2012, a total of 1,165 samples (91.4%) were from horses with record of JEV vaccination, of which 8 samples (0.7%) had VNT titers equal or above 1:640, which indicates JEV infection as designated by the criteria set for this study. In addition, of the 1,165 samples from horses that were vaccinated, 497 samples (42.7%) showed VNT titers below 1:10, indicating these horses were negative for antibodies to JEV. A total of 109 samples were from horses which were recorded as nonvaccinated for JEV, of which 5 (4.6%) were equal or above 1:160 (Table 3). For the 1,421 sera collected in 2013, a total of 1,127 samples (79.3%) were from horses with record of JEV vaccination, of which 13 samples (1.2%) had VNT titers equal or above 1:640, and 455 samples (40.4%) were below 1:10. A total of 294 samples were from horses with no
record of JEV vaccination, of which 36 (12.2%) were equal or above 1:160. 4. Discussion Global warming, growing urbanization, faster transportation, increased animal movements, and rapid spread of arthropod vectors all contribute to the continuous spreading of arboviruses into new geographic areas [3,15]. Many arboviruses were originally found in tropical regions such as Africa, South America, and in parts of Asia, but the geographic distribution and frequency of epidemic outbreaks of arboviral diseases have expanded dramatically across the world in the past several decades [23]. For example, in the case of EEE, which has been reported along the eastern coast of the United States and Canada, there have been an increase in the number of human EEE infections in the northernmost part of the outbreak areas [24]. Western equine encephalitis virus and VEEV have also been reported throughout most parts of the United States
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H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16 Table 2 Serologic test results for antibodies to Japanese encephalitis virus in horse sera.
Gyeonggi
Regions
Gangwon Seoul Incheon
Number of JEV-Positive Serum/Total Number of Serum (%) 2012
Chungbuk Chungnam Daejon
Jeonbuk
Gyeongbuk
Daegu Ulsan
Gyeongnam Gwangju
Busan
Jeonnam
Seoul Incheon Busan Daejeon Gwangju Daegu Ulsan Gyeonggi Gangwon Chungbuk Chungnam Jeonbuk Jeonnam Gyeongbuk Gyeongnam Jeju Total
164/277 0/2 165/199 3/3 6/7 5/5 d 135/363 6/8 11/12 10/11 61/91 9/13 7/11 1/1 154/271 737/1,274
2013 (59.2) (0) (82.9) (100) (85.7) (100) (37.1) (75) (91.7) (90.9) (67) (69.2) (63.6) (100) (56.8) (57.8)
d d 148/175 d d d 5/5 159/378 d d d 81/109 d 133/144 12/14 299/596 837/1,421
(84.6)
(100) (42.1)
(74.3) (92.4) (85.7) (50.2) (58.9)
Abbreviations: JEV, Japanese encephalitis virus; WNV, West Nile virus. For active surveillance, a total of 2,695 serum samples from clinically normal horses were collected from 2012 to 2013 throughout South Korea and serologically tested for WNV and JEV antibodies.
Jeju Fig. 1. Geographical regions of South Korea.
including Brazil and Argentina [25–28]. West Nile virus was first isolated in Africa but is now reported worldwide including the Middle East, Asia, Australia, and the United States [3]. Japanese encephalitis virus, which had been confined to countries in Asia, has since been reported in Australia [29]. For an arbovirus transmission cycle to become established, three factors are essential, which are the arbovirus, arthropod, and the vertebrate host, and if all factors are available in sufficient numbers, the virus will continuously circulate between the arthropod vector and competent host [30,31]. In South Korea, several species of mosquitoes including Culex spp. and Aedes spp. with the ability to transmit these arboviruses have been identified [6,32]. In addition, the possibility of new introduction of arboviruses such as EEEV, WEEV, VEEV, WNV, and SLEV into South Korea continues to grow due to climate change, increase in international movement of commodities and animal movements, and migratory birds [5]. Should these arboviruses become introduced into South Korea, it is possible that they could become quickly established due to the presence of viable vectors and hosts. In addition, because large proportion of the hosts would be naive to the viruses, huge economic losses could be expected due to large number of clinical cases. Therefore, it is important that South Korea conducts surveillance for arboviruses, focusing on viruses belonging to the families Togaviridae (genus Alphavirus) and Flaviviridae (genus Flavivirus), as these viruses are zoonotic and are associated with encephalitis, often with high mortality in humans as well as horses [2,33]. For these reasons, the present study
consisting of passive and active surveillance was conducted to determine the situation of major encephalitis-causing arboviruses in South Korean horses. No evidence of EEEV, WEEV, VEEV, WNV, and SLEV was found by passive surveillance, which supported the view that South Korea is free from these diseases. South Korea is endemic for JEV, and through passive surveillance conducted in this study, a JEV-positive horse was diagnosed. In addition, an active surveillance of healthy horses was conducted as part of the study, and 2,695 horses from across the country were serologically tested for WNV and JEV antibodies. For WNV serosurveillance, WNV ID Screen West Nile IgM antibody capture ELISA (ID VET, France) was used, which has a narrower window of detection compared with tests that detect IgG antibodies. However, for South Korea, which is not only endemic for JEV but also vaccinates horses for JEV, tests that detect IgG antibodies such as IgG ELISA were inappropriate due to cross-reactivity with JEV antibodies. Because horses in South Korea are regularly vaccinated to prevent infection of JEV [8,9,34], for the purpose of this study, a criteria was established to determine the JEV infection status based on the VN titers for vaccinated and nonvaccinated horses in South Korea. A total of 2,292 horses (85%) had a record of JEV vaccination, and 21 (0.9%) of these horses were positive for JEV infection, suggesting that they were infected with JEV but have since recovered or the infection was subclinical, perhaps due to protective immunity conferred by vaccination. Interestingly, of the vaccinated horses, 952 (41.5%) were JEV antibody negative, suggesting that a large percentage of the vaccinated horses had not seroconverted and that the current vaccination strategy for JEV may need to be reviewed. A total of 403 samples were from horses which were recorded as nonvaccinated for JEV, and 41 (10.2%) of these horses that had VN titers equal or above 1:160 were likely to have been infected with JEV, whereas 193 horses (47.9%) with VN titers between 1:10 and 1:80,
H.-Y. Jeoung et al. / Journal of Equine Veterinary Science 37 (2016) 11–16
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Table 3 Antibody titers against JEV by virus neutralization test in South Korean horses. Year
JEV Vaccination
Total No. of Samples
JE Antibody Titers <10
10
20
40
80
160
320
640
>640
2012
Vaccinated Nonvaccinated Total Vaccinated Nonvaccinated Total
1,165 109 1,274 1,127 294 1,421
497 40 537 455 129 584
148 14 162 124 26 150
164 18 182 135 36 171
153 18 171 150 27 177
110 14 124 117 40 157
66 2 68 82 21 103
19 2 21 51 12 63
7 1 8 10 3 13
1 0 1 3 0 3
2013
Abbreviation: JEV, Japanese encephalitis virus.
which were designated as being suspect could be considered to be due to infection with JEV but may also be due to nonspecific reaction to unknown factors. Another confounding factor that could not be overlooked was the possibility that some of the horses recorded as nonvaccinated had in fact been vaccinated but was not recorded as such, which would account for the presence of JEV antibodies in these horses. Future surveillance with a more strict recording system will be needed to better understand the JEV situation in South Korea. 5. Conclusions In this study, a surveillance of arbovirus-associated equine encephalitis was conducted, which could provide supporting evidence to demonstrate freedom from major arboviral diseases and was able to detect encephalitiscausing arboviral infection in horses such as JEV infection. Serologic surveillance demonstrated the possible presence of subclinical infection of JEV in the equine population and identified the need to review the current JEV vaccination strategy. Continued surveillance will be required to maintain the ability to quickly detect any new introductions and to provide information on the situation of equine encephalitis in South Korea. Acknowledgments This work was supported by a grant (Project Code No. P1541782-2012-12-02) from the Animal and Plant Quarantine Agency and Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and the authors are grateful to the National park research institute migratory bird center for their support in collection of dead wild birds. References [1] Keane DP, Little PB. Equine viral encephalomyelitis in Canada: a review of known and potential causes. Can Vet J 1987;28:497–504. [2] Davis LE, Beckham JD, Tyler KL. North American encephalitic arboviruses. Neurol Clin 2008;26:727–57. ix. [3] Go YY, Balasuriya UB, Lee CK. Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses. Clin Exp Vaccine Res 2014;3:58–77. [4] Spinsanti LI, Diaz LA, Glatstein N, Arselan S, Morales MA, Farias AA. Human outbreak of St. Louis encephalitis detected in Argentina, 2005. J Clin Virol 2008;42:27–33. [5] Yeh JY, Park JY, Ostlund EN. Serologic evidence of West Nile Virus in wild ducks captured in major inland resting sites for migratory waterfowl in South Korea. Vet Microbiol 2011;154:96–103.
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