• View in gallery

    IgG antibody response of flying foxes exposed to Japanese encephalitis virus (JEV) by infected mosquitoes, as detected by enzyme-linked immunosorbent assay (ELISA).

  • View in gallery

    IgG antibody response of flying foxes exposed to Japanese encephalitis virus (JEV) by inoculation, as detected by enzyme-linked immunosorbent assay (ELISA).

  • 1

    Mackenzie JS, Williams DT, Smith DW, 2007. Japanese encephalitis virus: the geographic distribution, incidence, and spread of a virus with a propensity to emerge in new areas. E. Tabor, ed. Emerging Viruses in Human Populations. Amsterdam: Elsevier, 201–268.

  • 2

    Mackenzie JS, Johansen CA, Ritchie SA, van den Hurk AF, Hall RA, 2002. Japanese encephalitis as an emerging virus: the emergence and spread of Japanese encephalitis virus in Australasia. Curr Top Microbiol Immunol 267 :49–73.

    • Search Google Scholar
    • Export Citation
  • 3

    Hanna JN, Ritchie SA, Phillips DA, Shield J, Bailey MC, Mackenzie JS, Poidinger M, McCall BJ, Mills PJ, 1996. An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 165 :256–260.

    • Search Google Scholar
    • Export Citation
  • 4

    Hanna JN, Ritchie SA, Phillips DA, Lee JM, Hills SL, van den Hurk AF, Pyke AT, Johansen CA, Mackenzie JS, 1999. Japanese encephalitis in north Queensland, Australia, 1998. Med J Aust 170 :533–536.

    • Search Google Scholar
    • Export Citation
  • 5

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 6

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, Mackenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 7

    Buescher EL, Scherer WF, Rosenberg MZ, McClure HE, 1959. Immunologic studies of Japanese encephalitis virus in Japan. III. Infection and antibody responses of birds. J Immunol 83 :605–613.

    • Search Google Scholar
    • Export Citation
  • 8

    Buescher EL, Scherer WF, McClure HE, Moyer JT, Rosenberg MZ, Yoshii M, Okada Y, 1959. Ecologic studies of Japanese encephalitis virus in Japan. IV. Avian infection. Am J Trop Med Hyg 8 :678–688.

    • Search Google Scholar
    • Export Citation
  • 9

    Gresser I, Hardy JL, Hu SMK, Scherer WF, 1958. Factors influencing transmission of Japanese B encephalitis virus by a colonized strain of Culex tritaeniorhynchus Giles, from infected pigs and chicks to susceptible pigs and birds. Am J Trop Med Hyg 7 :365–373.

    • Search Google Scholar
    • Export Citation
  • 10

    Scherer WF, Moyer JT, Izumi T, Gresser I, McCown J, 1959. Ecologic studies of Japanese encephalitis virus in Japan. VI. Swine infection. Am J Trop Med Hyg 8 :698–706.

    • Search Google Scholar
    • Export Citation
  • 11

    Endy TP, Nisalak A, 2002. Japanese encephalitis virus: ecology and epidemiology. Curr Top Microbiol Immunol 267 :11–48.

  • 12

    Mackenzie JS, Field HE, Guyatt KJ, 2003. Managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and Australian bat lyssavirus. J Appl Microbiol 94 :59S–69S.

    • Search Google Scholar
    • Export Citation
  • 13

    Sulkin SE, Allen R, 1974. Virus infections in bats. Melnick JL, ed. Monographs in Virology, Vol. 8. Basel: S. Karger, 1–103.

  • 14

    Banerjee K, Ilkal MA, Bhat HR, Sreenivasan MA, 1979. Experimental viraemia with Japanese encephalitis virus in certain domestic and peridomestic vertebrates. Indian J Med Res 70 :364–368.

    • Search Google Scholar
    • Export Citation
  • 15

    Banerjee K, Ilkal MA, Deshmukh PK, 1984. Susceptibility of Cynopterus sphinx (frugivorus bat) and Suncus murinus (house shrew) to Japanese encephalitis virus. Indian J Med Res 79 :8–12.

    • Search Google Scholar
    • Export Citation
  • 16

    Webb NJ, Tidemann CR, 1996. Mobility of Australian flying foxes, Pteropus spp. (Megachiroptera): evidence from genetic variation. Proc R Soc Lond B Biol Sci 263 :497–502.

    • Search Google Scholar
    • Export Citation
  • 17

    Jonsson NN, Johnston SD, Field H, de Jong C, Smith C, 2004. Field anaesthesia of three Australian species of flying fox. Vet Rec 154 :664.

    • Search Google Scholar
    • Export Citation
  • 18

    Beaty BJ, Calisher CH, Shope RE, 1995. Arboviruses. Lennette EH, Lennette DA, Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. Washington, DC: American Public Health Association, 189–212.

  • 19

    Halpin K, Young PL, Field HE, Mackenzie JS, 2000. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 81 :1927–1932.

    • Search Google Scholar
    • Export Citation
  • 20

    Crameri G, Wang LF, Morrissy C, White J, Eaton BT, 2002. A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies. J Virol Methods 99 :41–51.

    • Search Google Scholar
    • Export Citation
  • 21

    Goddard LB, Roth AE, Reisen WK, Scott TW, 2002. Vector competence of California mosquitoes for West Nile virus. Emerg Infect Dis 8 :1385–1391.

    • Search Google Scholar
    • Export Citation
  • 22

    Turell MJ, Kay BH, 1998. Susceptibility of selected strains of Australian mosquitoes (Diptera: Culicidae) to Rift Valley fever virus. J Med Entomol 35 :132–135.

    • Search Google Scholar
    • Export Citation
  • 23

    Pyke AT, Smith IL, van den Hurk AF, Northill JA, Chuan TF, Westacott AJ, Smith GA, 2004. Detection of Australasian flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays. J Virol Methods 117 :161–167.

    • Search Google Scholar
    • Export Citation
  • 24

    Biggerstaff BJ, 2003. PooledInfRate: a Microsoft Excel add-in to compute prevalence estimates from pooled samples. Fort Collins, CO: Centers for Disease Control and Prevention.

  • 25

    Smith IL, Westacott AJ, Smith GA, 2005. Development and implementation of a porcine IgM capture enzyme linked immunosorbent assay for the detection of Japanese encephalitis virus antibodies in sentinel pigs. Arbovirus Res Aus 9 :352–356.

    • Search Google Scholar
    • Export Citation
  • 26

    Pyke AT, Phillips DA, Chuan TF, Smith GA, 2004. Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine. BMC Microbiol 4 :3–10.

    • Search Google Scholar
    • Export Citation
  • 27

    Johnsen DO, Edelman R, Grossman RA, Muangman D, Pomsdhit J, Gould DJ, 1974. Study of Japanese encephalitis virus in Chiangmai Valley, Thailand. V. Animal infections. Am J Epidemiol 100 :57–68.

    • Search Google Scholar
    • Export Citation
  • 28

    Oda K, Igarashi A, Keong CT, Hong CC, Vijayamalar B, Sinniah M, Hassan SS, Tanaka H, 1996. Cross-sectional serosurvey for Japanese encephalitis specific antibody from animal sera in Malaysia 1993. Southeast Asian J Trop Med Public Health 27 :463–470.

    • Search Google Scholar
    • Export Citation
  • 29

    Takashima I, Watanabe T, Ouchi N, Hashimoto N, 1988. Ecologic studies of Japanese encephalitis virus in Hokkaido: interepidemic outbreaks of swine abortion and evidence for the virus to overwinter locally. Am J Trop Med Hyg 38 :420–427.

    • Search Google Scholar
    • Export Citation
  • 30

    Burns KF, 1950. Congenital Japanese B encephalitis infection of swine. Proc Soc Exp Biol Med 75 :621–625.

  • 31

    Lord CC, Rutledge CR, Tabachnick WJ, 2006. Relationships between host viremia and vector susceptibility for arboviruses. J Med Entomol 43 :623–630.

    • Search Google Scholar
    • Export Citation
  • 32

    Gould DJ, Byrne RJ, Hayes DE, 1964. Experimental infection of horses with Japanese encephalitis virus by mosquito bite. Am J Trop Med Hyg 13 :742–746.

    • Search Google Scholar
    • Export Citation
  • 33

    Kay BH, Young PL, Hall RA, Fanning ID, 1985. Experimental infection with Murray Valley encephalitis virus. Pigs, cattle, sheep, dogs, rabbits, macropods and chickens. Aust J Exp Biol Med Sci 63 :109–126.

    • Search Google Scholar
    • Export Citation
  • 34

    Gómez A, Kramer LD, Dupuis AP, Kilpatrick AM, Davis LJ, Jones MJ, Daszak P, Aguirre AA, 2008. Experimental infection of eastern gray squirrels (Sciurus carolinensis) with West Nile virus. Am J Trop Med Hyg 79 :447–451.

    • Search Google Scholar
    • Export Citation
  • 35

    Brown AN, Kent KA, Bennett CJ, Bernard KA, 2007. Tissue tropism and neuroinvasion of West Nile virus do not differ for two mouse strains with different survival rates. Virology 368 :422–430.

    • Search Google Scholar
    • Export Citation
  • 36

    Turell MJ, Tammariello RF, Spielman A, 1995. Nonvascular delivery of St. Louis encephalitis and Venezuelan equine encephalitis viruses by infected mosquitoes (Diptera: Culicidae) feeding on a vertebrate host. J Med Entomol 32 :563–568.

    • Search Google Scholar
    • Export Citation
  • 37

    Turell MJ, Spielman A, 1992. Nonvascular delivery of Rift Valley fever virus by infected mosquitoes. Am J Trop Med Hyg 47 :190–194.

  • 38

    Higgs S, Schneider BS, Vanlandingham DL, Klingler KA, Gould EA, 2005. Nonviremic transmission of West Nile virus. Proc Natl Acad Sci USA 102 :8871–8874.

    • Search Google Scholar
    • Export Citation
  • 39

    McGee CE, Schneider BS, Girard YA, Vanlandingham DL, Higgs S, 2007. Nonviremic transmission of West Nile virus: evaluation of the effects of space, time, and mosquito species. Am J Trop Med Hyg 76 :424–430.

    • Search Google Scholar
    • Export Citation
  • 40

    Markus N, Hall L, 2004. Foraging behaviour of the black flying fox (Pteropus alecto) in the urban landscape of Brisbane, Queensland. Wildl Res 31 :345–355.

    • Search Google Scholar
    • Export Citation
  • 41

    Breed AC, Smith CS, Epstein JH, 2006. Winged wanderers: long distance movements of flying foxes. MacDonald DW, ed. The Encyclopedia of Mammals. Oxford: Oxford University Press, 474–475.

  • 42

    Ritchie SA, Rochester W, 2001. Wind-blown mosquitoes and introduction of Japanese encephalitis into Australia. Emerg Infect Dis 7 :900–903.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Transmission of Japanese Encephalitis Virus from the Black Flying Fox, Pteropus alecto, to Culex annulirostris Mosquitoes, Despite the Absence of Detectable Viremia

View More View Less
  • 1 Virology, Queensland Health Forensic and Scientific Services, Coopers Plains, Queensland, Australia; School of Chemical and Molecular Biosciences, University of Queensland, St Lucia, Queensland, Australia; Department of Primary Industries and Fisheries, Animal Research Institute, Yeerongpilly, Queensland, Australia; Australian Army Malaria Institute, Gallipoli Barracks, Enoggera, Queensland, Australia; Australian Biosecurity Cooperative Research Centre, Curtin University of Technology, Perth, Western Australia, Australia

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.

INTRODUCTION

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus of Southeast Asia responsible for over 40,000 clinical cases annually, with a 25% fatality rate.1 Japanese encephalitis virus is an emerging virus in the Australasian region with potentially serious public and animal health implications.2 Widespread JEV activity was first detected in Australia in 1995 on Badu Island in Torres Strait, when it caused an outbreak consisting of three human cases and two deaths.3 Subsequently, except for 1999, JEV activity was recognized every year in the Torres Strait between 1995 and 2005, when the sentinel pig program was discontinued. In 1998, human and pig infections were identified for the first time on the Australian mainland at the mouth of the Mitchell River on Cape York Peninsula.4 Entomologic studies implicated Culex annulirostris as the primary mosquito vector of JEV in northern Australia. 5,6

Pigs and ardeid wading birds, such as herons and egrets are the primary amplifying hosts of JEV, as they produce viremia sufficient to infect mosquitoes.710 Although humans and horses can develop fatal encephalitis, they are considered to be dead-end hosts of the virus, developing only low-level viremia.11

Should JEV become established on the Australian mainland, the potential importance of Australian fauna species acting as vertebrate amplifying hosts is largely unknown. Experimental studies at the Commonwealth Scientific and Industrial Research Organization Australian Animal Health Laboratory previously examined the possible role of a number of Australian native species (such as wallabies and possums) as reservoir or amplifying hosts.2 However, flying foxes (Megachiroptera: Pteropididae) were not included in these studies, and mosquitoes were not used as the route of virus exposure or to show transmission of JEV. Flying foxes are of particular interest as a potential reservoir or amplifying host of JEV as a result of their nomadic behavior, and because of their increasing abundance in major urban areas in eastern Australia. Certainly, the behavioral ecology of flying foxes deems them potential dispersal hosts of mammalian viruses, and several recently emerged zoonotic viruses, including Hendra virus (HeV) and Australian bat lyssavirus, have been described in flying foxes in Australia. 12 Furthermore, both experimental and natural infection studies have suggested a possible role for bats in the transmission cycles of a number of arboviruses, including JEV (reviewed by Sulkin and Allen).13 However, the majority of these experiments have examined insectivorous bats of the order Microchiroptera. The only experimental infection of Megachiropteran bats with JEV has been undertaken in India. In these earlier studies, viremia developed in both Rousettus leschenaulti and Cynopterus sphinx, with the latter species able to infect recipient mosquitoes. 14,15

We undertook laboratory-based infection and transmission experiments to determine whether flying foxes can act as amplifying hosts for JEV. Specifically, we examined 1) the ability of flying foxes to develop clinical illness, viremia, and specific antibodies after being exposed to JEV via infected mosquitoes or by inoculation; and 2) the likelihood of recipient mosquitoes becoming infected after feeding on flying foxes that had been previously exposed to JEV. The Black flying fox, Pteropus alecto, was chosen because of its widespread geographic distribution throughout northern Australia, including areas where JEV has previously occurred. 16

MATERIALS AND METHODS

All animal experimentation complied with the Queensland Animal Care and Protection Act 2001 and was approved by the Queensland Department of Primary Industries Animal Ethics Committee (AEC; approval no. 33/08/02), University of Queensland AEC (approval no. MICRO/PARA/463/02/), and Queensland Health Forensic and Scientific Services AEC (QHFSS; approval no. VIR 1/03/32). In addition, the collection of wild flying foxes was approved by the Queensland Parks and Wildlife Service (Scientific Purposes permit no. WISP01419503). As JEV is considered an exotic virus in Australia, all infection and transmission experiments were undertaken at the Physical Containment Level 3 insectary/animal house at QHFSS, Brisbane.

Animals.

Pteropus alecto were collected from flying fox colonies located in the Brisbane suburbs of East Brisbane (27°28′S, 153°03′E) and Indooroopilly (27°30′S, 152°58′E), which also contained the Grey-headed flying fox, Pteropus poliocephalus and the Little red flying fox, Pteropus scapulatus. Flying foxes returning from overnight foraging were captured using mist nets for a period of approximately 90 min before sunrise. Flying foxes were anaesthetized using the inhalation anesthetic Isoflurane (Isoflurane Inhalation Anesthetic, Laser Animal Health, Salisbury, Australia) administered by a portable Ohmeda Isotec 3 vaporizer (BOC Health Care, West Yorkshire, England) at a concentration of 5% for induction and 1.5% during maintenance with an oxygen flow rate of 1 L/min. 17 Each bat was assessed for age, weight, forearm length, and female bats for pregnancy status by abdomen palpitation. Pregnant females were excluded from the study and released. To aid in future identification, each flying fox was injected with a microchip containing a unique identification number (LifeChip, Destron Fearing, South St. Paul, MN). A 1–2 mL blood sample was obtained from the propatagial vein using a 3 mL syringe (Terumo [Philippines] Corporation, Laguna, Philippines), a 0.50 × 16 mm needle (Terumo [Philippines] Corporation), and lithium heparin (Heparin Injection BP, David Bull Laboratories, Mulgrave North, Australia) as an anticoagulant. The serum fraction was tested for JEV-specific antibodies using a neutralization assay. 18 As HeV circulates within wild flying fox populations in Brisbane, 19 the serum samples also were tested for antibodies to this virus, using the HeV Serum Neutralization Test developed and performed at the CSIRO Australian Animal Health Laboratory, Geelong. 20

Flying foxes were held at the Queensland Department of Primary Industries and Fisheries, Animal Research Institute (ARI), Yeerongpilly, for ≤ 14 d to allow for a second serum sample to be obtained for the completion of baseline serology. Any animals showing evidence of current or previous infection with either JEV or HeV were excluded from the study.

Virus strain.

All experiments used the JEV TS3306 strain isolated from Aedes vigilax collected from Badu Island in February 1998. The virus had been passaged once in C6/36 (Ae. albopictus salivary gland) cells and twice in porcine stable-equine kidney (PS-EK) cells. The titer of virus stock was 108 PS-EK TCID50 (tissue culture infectious dose)50/mL.

Mosquito strain.

The mosquitoes used for all infection and transmission studies were Culex annulirostris from colonies housed at the Australian Army Malaria Institute, Brisbane. The Cx. annulirostris colony was established from adults collected from the Boondall Wetlands, Brisbane in 1998. Vector competence experiments had previously showed that this strain of Cx. annulirostris had infection and transmission rates of 100% and 83%, respectively, for the same strain of JEV used in the current study.6

Infection of mosquitoes with JEV.

The 2- to 4-day-old mosquitoes were starved for 24 hr before feeding for 30 min to 1 hr on a virus suspension using the hanging drop method of Goddard and others. 21 The virus suspension contained JEV stock diluted in heparinized rabbit blood and 1% sucrose. Two to three milliliter (mL) of this suspension (first heated to 37 ± 1.0°C) was dropped onto the gauze covering the open end of 700 mL plastic containers that housed the mosquitoes. To determine the virus titer of the blood meal at the commencement and cessation of feeding, samples of the blood/virus suspension were diluted 1:20 in growth media (GM; Opti-MEM [Invitrogen, Grand Island, NY] with 3% fetal bovine serum, antibiotics and fungizone) and stored at −70°C for later titration.

After 18 hr, mosquitoes were anesthetized with CO2 and blood engorged mosquitoes were placed into 1 L containers within an environmental growth cabinet (Sanyo Electric, Gunma, Japan) maintained at 28°C, 70–75% RH, and 12:12 (L:D), and offered 10% sucrose as a nutrient source. Mosquitoes were held under such conditions for an extrinsic incubation period of 13 d. To determine if the mosquitoes intended for the infection of the flying foxes were indeed infected, a sample of ≤ 6 individual mosquitoes were removed on day 12 post virus exposure and tested for the presence of virus.

Flying fox husbandry.

On the afternoon before the infection experiments, flying foxes were moved from ARI to QHFSS and placed in individual cages in a room within the PC3 animal house. The dimensions of the cages were 900 × 900 × 900 mm, and were of wire mesh construction (25 × 25 × 25 mm aperture), elevated 1 m above the ground, with 10–15 cm between cages. Seasonal fruits (including rockmelon, bananas, mangoes, apples, etc.), supplemented by full cream milk powder, were fed to the flying foxes between 4 and 6 pm daily; water was provided ad libatum via drip bottles. Artificial light was provided for the flying foxes each day between the hours of usual sunrise and sunset.

Exposure of flying foxes to JEV.

Individual flying foxes were restrained, placed in calico bags, and transferred from the animal room to the insectary, located within the QHFSS PC3 insectary/animal house. Before infection and transmission procedures, flying foxes were anesthetized as described previously. Ten flying foxes were exposed to JEV via mosquito bite, during two separate trials, each involving five flying foxes. Fifteen donor mosquitoes (previously exposed to JEV), in 30 × 40 mm containers with gauze covering the open end, were allowed to feed on the upper leg for ≤ 15 min. At the same time, 0.5 mL blood samples were obtained from the propatagial vein and 50 μL diluted 1/10 in GM for virus detection. The serum fraction of the remaining 450 μL was retained for serology. Animals were also weighed and rectal temperatures obtained during each flying fox/mosquito interaction. After recovering from the anesthesia (usually within 5 min), flying foxes were returned to their cages. To determine the virus titer of donor mosquitoes at the time of exposure, mosquitoes were killed with CO2 and blood engorged mosquitoes stored at −70°C to await analysis. Five additional animals were exposed to JEV by being inoculated subcutaneously in the upper thigh area with 0.1 mL of 104.8 PS-EK TCID50/mL of virus.

Flying foxes were observed at least twice daily for clinical symptoms of encephalitis (including fever, reduced appetite, ruffled fur, decreased grooming, increased vocalization, ataxia, eye closure, muscular rigidity, etc.) caused by infection with JEV.

To determine if infected bats could produce a viremia capable of infecting mosquitoes, batches of recipient mosquitoes were allowed to feed on the upper leg, interfemoral membrane, or the plagiopatagium of each flying fox on days 1–5, 7, 9, 14, 20/21, 23/28 post exposure for the mosquito-exposed animals, and days 3–5 for the inoculated animals. Because of unforseen external circumstances independent of this study, the first series of infections were concluded on day 23 instead of day 28 post exposure. During each mosquito/flying fox interaction, blood samples, temperatures, and weights for each animal were obtained while the animal was anaesthetized. Batches of recipient mosquitoes were maintained for 10 d as described previously, before being killed with CO2 and frozen at −70°C to await virus assay.

At the conclusion of the experiments, flying foxes were euthanized while under anesthesia with a lethal intravenous injection of Pentobarbitone sodium (Lethabarb Euthenasia Injection; Virbac [Australia] Pty. Ltd., Peakhurst, Australia). The carcasses were necropsied, during which the brain, heart, liver, lung, spleen, and kidneys removed and retained, before autoclaving and disposal.

Virus assay.

The blood/virus mixture used for mosquito infection was titrated as 10-fold dilutions in a 96-well microtiter plate containing confluent PS-EK cell monolayers. Plates were incubated at 37°C with 5% CO2 and checked daily for any cytopathic effect (CPE) for ≤ 7 d. The amount of virus ingested by individual mosquitoes was estimated based on an average blood meal volume of approximately 3 μL. 22 The heads and bodies of the representative sample of donor mosquitoes were homogenized separately in 1 mL of GM using a SPEX 8000 mixer/mill (Spex Industries, Edison, NJ). Viral RNA was extracted using the QIAamp Viral RNA kit (Qiagen, Clifton Hill, Australia) before being tested for JEV using a real time TaqMan reverse transcriptase-polymerase chain reaction procedure (RT-PCR). 23 In this assay, a cycle threshold (C t ) value of 40 corresponded to < 0.001 of one plaque-forming unit (PFU) of virus, and was deemed to be not detected. To determine viral titers of donor mosquitoes, whole individual mosquitoes were homogenized in 1mL of tissue culture medium and filtered using a 0.2 μm filter, before being titrated as 10-fold dilutions on confluent PS-EK cell monolayers. Recipient mosquitoes were pooled in batches of ≤ 12 according to bat number and day post exposure. Pools of recipient mosquitoes were then homogenized in 1 mL of GM. Viral RNA was extracted from the supernatant using the QIAamp Viral RNA kit before being tested using the TaqMan RT-PCR. Infection rates were calculated for the pools of recipient mosquitoes using the PooledInfRate statistical software package.24

To test for viremia, all blood samples collected from the first five flying foxes exposed to JEV by mosquito bite, were initially titrated as serial 10-fold dilutions on PS-EK monolayers as described previously. When virus was not detected using the cell culture system, the blood samples from this trial and from the remaining flying foxes exposed to JEV by mosquito bite and by inoculation were screened using the TaqMan RT-PCR. Any positive blood samples in the RT-PCR were titrated as 10-fold dilutions on PS-EK cell monolayers as described above.

Serology.

To establish if seroconversion to JEV had occurred in flying foxes after JEV exposure, an enzyme-linked immunosorbent assay (ELISA) was developed using peroxidase conjugated Protein A/G to monitor IgG response. Porcine sera, which had previously been shown to contain JEV-specific antibodies, were used for assay optimization and as positive controls. 25 Negative controls consisted of porcine sera, which had previously tested negative for flavivirus antibodies. The ELISA was performed using Maxisorp strips (NUNC A/S, Roskilde, Denmark), which were coated with inactivated JEV antigen in carbonate buffer (pH 9.6). 26 After overnight incubation at 4°C, the strips were washed five times in phosphate buffered saline (PBS)-Tween. Sera collected from the 15 flying foxes during the experiments were added at a 1/100 dilution to the wells and incubated for 1 hr at 37°C before a second wash. The presence of JEV-specific antibody was detected by the addition of ImmunoPure Protein A/G–peroxidase conjugate (Pierce, Rockford, IL) at a dilution of 1/160,000. The conjugate was incubated for 1 hr at 37°C. The strips were again washed before the addition of KBlue substrate (Neogen, Lexington, KY) for 10 min. The reaction was stopped by the addition of 1N H2SO4. Absorbances were measured at 450 nm with a reference wavelength of 650 nm.

RESULTS

Virus titer of donor mosquitoes.

After 13 d extrinsic incubation, 1–7 infected donor mosquitoes fed on each of the flying foxes. The virus titer of donor mosquitoes ranged between 101.0 and 106.1 TCID50 per mosquito (Table 1).

Clinical symptoms of JEV infection in the flying foxes.

None of the flying foxes displayed any clinical symptoms of encephalitis consistent with infection by JEV.

Viremia.

None of the flying foxes exposed to JEV by mosquito bite developed a detectable viremia. Only one inoculated flying fox (microchip no. 3003) developed a viremia on the fourth day post inoculation. However, this viremia was of a low level, as evidenced by the high cycle threshold score of 36 cycles in the TaqMan RT-PCR and a lack of detectable viral replication when inoculated onto PS-EK cells.

Infection of recipient mosquitoes.

A total of 2,464 recipient mosquitoes in 305 pools were processed for detection of JEV RNA. Of these mosquitoes, 2,191 had fed on the flying foxes that had been infected by mosquito bite and 273 on the inoculated flying foxes. JEV RNA was detected in 7 pools of recipient mosquitoes that had fed on 4 flying foxes, indicating virus transmission (Table 2). Recipient mosquitoes were infected on either Days 3, 4, or 5 post exposure and infection rates for these pools ranged between 38.5 and 90.9 per 1,000 mosquitoes.

Antibody response to infection.

Sixty percent (6/10) of the flying foxes that were exposed to JEV via mosquito had detectable IgG responses in the Protein A/G ELISA, indicating a seroconversion to JEV (Figure 1). In the experiments using inoculation as the source of infection, 100% of the flying foxes seroconverted to JEV (Figure 2).

DISCUSSION

As shown by serologic studies, numerous animals are naturally infected with JEV, 27,28 although humans and horses are the only animals that develop severe and fatal disease, characterized by acute encephalitis. In some instances, infection in pigs can result in abortion and stillbirth in sows, and reduced sperm production in boars. 29,30 In the current study, infection with JEV did not result in overt clinical illness in any of the flying foxes. These results support previous studies of both Microchiropteran and Megachiropteran bats, in which experimental infection with JEV failed to produce any signs of encephalitis, even if inoculated directly into the brain. 1315

Common opinion considers that virus circulating in the blood is essential to facilitate ingestion by a mosquito and that a threshold level of viremia must be reached before infections of vectors can occur. 31 Therefore, infection of recipient mosquitoes after feeding on flying foxes without detectable viremia was unexpected. However, this is not a novel phenomenon, as earlier experiments have shown that recipient mosquitoes can be infected after feeding on horses and Agile wallabies (Macropus agilis) without detectable viremia that had previously been exposed to JEV and Murray Valley encephalitis virus, respectively. 32,33

In our study, we used a sensitive real-time RT-PCR, which is able to detect viral RNA from 0.005 PFU of virus. 23 Therefore, it should have been more likely to detect viral RNA than earlier methods of virus assay used in vertebrate studies. Importantly, the results of this study emphasize the need to use highly susceptible recipient arthropods to show transmission when assessing the relative ability of vertebrate species to serve as amplifying hosts of arboviruses.

This cryptic viremia may be explained by the replication of virus in skin or dendritic cells at the site of inoculation of the flying fox, with or without entering the bloodstream. In our experiments, some recipient mosquitoes fed at the same site (i.e., the upper leg) as the donor mosquitoes and may have subsequently imbibed virus liberated from the cells and tissues damaged by the action of the probing mouthparts. Indeed, West Nile virus (WNV), a member of the JEV serologic group, has been shown to replicate in the skin cells of eastern grey squirrels (Sciurus carolinensis ) and mice at the site of inoculation, with viral loads in the latter species increasing from an undetectable level on the first day post exposure to a titer of ≤ 105 PFU/g at day 7 post exposure. 34,35 Initial infection of skin tissue is possibly caused by the deposition of virions extravascularly in tissue surrounding capillaries and not directly into the vascular system while mosquitoes are probing and locating blood vessels. 36,37

This is a different concept to purported nonviremic transmission (NVT) of arboviruses, where infection of recipient mosquitoes with WNV occurs when they feed simultaneously with infected donor mosquitoes and, importantly, before viral replication in the host. 38 When McGee and others 39 assessed the effects of time and space on NVT, recipient mosquitoes could be infected ≤ 45 min after the donor mosquitoes had fed. However, in our experiments, transmission to recipient mosquitoes occurred 3 to 5 d after exposure, providing adequate time for the virus to replicate at the site of inoculation. Further experiments are required to elucidate whether recipient mosquitoes can be infected while probing cells and tissues before cannulation of the blood vessel.

Although infection rates in recipient mosquitoes were relatively low in this experiment, the large number of animals in P. alecto colonies, in which populations can reach tens of thousands, 40 may provide sufficient numbers of hosts to infect local mosquitoes. Indeed, large flying fox colonies are present on some Torres Strait islands where JEV activity has been observed, including Badu Island, the island that has yielded the majority of JEV isolates. Furthermore, analysis of mosquito host feeding patterns has revealed that Cx. annulirostris does feed on flying foxes on Badu Island (van den Hurk AF, Hall-Mendelin S, and Cheah WY, unpublished data).

Only a small number of infected flying foxes would be required to facilitate spillover of JEV into local bird and/or pig populations via mosquito bite and thus initiate epizootic activity. Importantly, because there is considerable movement of P. alecto between Cape York Peninsula, the Torres Strait, and Papua New Guinea, it is conceivable that flying foxes may have introduced JEV into northern Australia from the New Guinea landmass, as suggested by Mackenzie and others.2 Flying foxes can travel > 50 km in a single night, so that it would take approximately 2 and 4 d for a flying fox to traverse the distance between southern Papua New Guinea and Badu Island, or Cape York Peninsula, respectively. 41 This is well within the 3–5 d time period after JEV exposure that flying foxes were able to infect recipient mosquitoes in our experiments. However, whereas flying foxes undoubtedly undertake foraging flights to different islands, such sporadic activity could not account for the synchronicity of the widespread appearance of JEV on eastern Torres Strait islands. This phenomenon suggests an alternative mechanism of incursion, such as wind-blown mosquitoes, may be more likely. 42

Table 1

Number and virus titer of donor mosquitoes used to infect flying foxes

Table 1
Table 2

Infection rates (IRs) of pools of infected recipient mosquitoes that were allowed to feed on Japanese encephalitis virus (JEV) exposed flying foxes*

Table 2
Figure 1.
Figure 1.

IgG antibody response of flying foxes exposed to Japanese encephalitis virus (JEV) by infected mosquitoes, as detected by enzyme-linked immunosorbent assay (ELISA).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 3; 10.4269/ajtmh.2009.81.457

Figure 2.
Figure 2.

IgG antibody response of flying foxes exposed to Japanese encephalitis virus (JEV) by inoculation, as detected by enzyme-linked immunosorbent assay (ELISA).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 3; 10.4269/ajtmh.2009.81.457

*

Address correspondence to Andrew F. van den Hurk, Virology, Queensland Health Forensic and Scientific Services, 39 Kessels Rd, Coopers Plains, Queensland 4108, Australia. E-mail: andrew_hurk@health.qld.gov.au

Authors’ addresses: Andrew F. van den Hurk, Ina L. Smith, Judith A. Northill, Carmel T. Taylor, and Greg A. Smith, Virology, Queensland Health Forensic and Scientific Services, 39 Kessels Rd., Coopers Plains, Queensland 4108, Australia, Tel: 617-3274-9135, Fax: 617-3000-9186, E-mails: andrew_hurk@health.qld.gov.au, ina_smith@health.qld.gov.au, judy_northill@health.qld.gov.au, carmel_taylor@health.qld.gov.au, and greg_smith@health.qld.gov.au. Craig S. Smith and Hume E. Field, Department of Primary Industries and Fisheries, Animal Research Institute, 665 Fairfield Road, Yeerongpilly, Queensland 4105, Australia, E-mails: Craig.S.Smith@dpi.qld.gov.au and hume.field@dpi.qld.gov.au. Cassie C. Jansen, formerly Australian Army Malaria Institute, currently Australian Biosecurity Cooperative Research Centre, University of Queensland, St. Lucia, Queensland 4072, Australia, E-mail: cassie_jansen@health.qld.gov.au. John S. Mackenzie, Australian Biosecurity Cooperative Research Centre, Centre for International Health, Curtin University of Technology, GPO U1987, Perth, Western Australia, 6845, Australia, E-mail: J.Mackenzie@curtin.edu.au.

Acknowledgments: We thank Bruce Harrower for undertaking the post mortems on the flying foxes; Amanda McLaughlin, Carol de Jong, and Janine Barrett for assistance with flying fox collection; and Sadet Davis, Teck Chuan, Petrina Johnson, Donna Mackenzie, and Russell Simmons for technical assistance. We also thank Les Hall for discussions on flying fox movements in the Torres Strait and Laura Kramer, Scott Ritchie, and Roy Hall for their comments on the manuscript.

Financial support: This study was funded by the Australian Health Minister’s Advisory Council (AHMAC) Priority Driven Research Program and Queensland Health.

REFERENCES

  • 1

    Mackenzie JS, Williams DT, Smith DW, 2007. Japanese encephalitis virus: the geographic distribution, incidence, and spread of a virus with a propensity to emerge in new areas. E. Tabor, ed. Emerging Viruses in Human Populations. Amsterdam: Elsevier, 201–268.

  • 2

    Mackenzie JS, Johansen CA, Ritchie SA, van den Hurk AF, Hall RA, 2002. Japanese encephalitis as an emerging virus: the emergence and spread of Japanese encephalitis virus in Australasia. Curr Top Microbiol Immunol 267 :49–73.

    • Search Google Scholar
    • Export Citation
  • 3

    Hanna JN, Ritchie SA, Phillips DA, Shield J, Bailey MC, Mackenzie JS, Poidinger M, McCall BJ, Mills PJ, 1996. An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 165 :256–260.

    • Search Google Scholar
    • Export Citation
  • 4

    Hanna JN, Ritchie SA, Phillips DA, Lee JM, Hills SL, van den Hurk AF, Pyke AT, Johansen CA, Mackenzie JS, 1999. Japanese encephalitis in north Queensland, Australia, 1998. Med J Aust 170 :533–536.

    • Search Google Scholar
    • Export Citation
  • 5

    Ritchie SA, Phillips D, Broom A, Mackenzie J, Poidinger M, van den Hurk A, 1997. Isolation of Japanese encephalitis virus from Culex annulirostris in Australia. Am J Trop Med Hyg 56 :80–84.

    • Search Google Scholar
    • Export Citation
  • 6

    van den Hurk AF, Nisbet DJ, Hall RA, Kay BH, Mackenzie JS, Ritchie SA, 2003. Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus. J Med Entomol 40 :82–90.

    • Search Google Scholar
    • Export Citation
  • 7

    Buescher EL, Scherer WF, Rosenberg MZ, McClure HE, 1959. Immunologic studies of Japanese encephalitis virus in Japan. III. Infection and antibody responses of birds. J Immunol 83 :605–613.

    • Search Google Scholar
    • Export Citation
  • 8

    Buescher EL, Scherer WF, McClure HE, Moyer JT, Rosenberg MZ, Yoshii M, Okada Y, 1959. Ecologic studies of Japanese encephalitis virus in Japan. IV. Avian infection. Am J Trop Med Hyg 8 :678–688.

    • Search Google Scholar
    • Export Citation
  • 9

    Gresser I, Hardy JL, Hu SMK, Scherer WF, 1958. Factors influencing transmission of Japanese B encephalitis virus by a colonized strain of Culex tritaeniorhynchus Giles, from infected pigs and chicks to susceptible pigs and birds. Am J Trop Med Hyg 7 :365–373.

    • Search Google Scholar
    • Export Citation
  • 10

    Scherer WF, Moyer JT, Izumi T, Gresser I, McCown J, 1959. Ecologic studies of Japanese encephalitis virus in Japan. VI. Swine infection. Am J Trop Med Hyg 8 :698–706.

    • Search Google Scholar
    • Export Citation
  • 11

    Endy TP, Nisalak A, 2002. Japanese encephalitis virus: ecology and epidemiology. Curr Top Microbiol Immunol 267 :11–48.

  • 12

    Mackenzie JS, Field HE, Guyatt KJ, 2003. Managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and Australian bat lyssavirus. J Appl Microbiol 94 :59S–69S.

    • Search Google Scholar
    • Export Citation
  • 13

    Sulkin SE, Allen R, 1974. Virus infections in bats. Melnick JL, ed. Monographs in Virology, Vol. 8. Basel: S. Karger, 1–103.

  • 14

    Banerjee K, Ilkal MA, Bhat HR, Sreenivasan MA, 1979. Experimental viraemia with Japanese encephalitis virus in certain domestic and peridomestic vertebrates. Indian J Med Res 70 :364–368.

    • Search Google Scholar
    • Export Citation
  • 15

    Banerjee K, Ilkal MA, Deshmukh PK, 1984. Susceptibility of Cynopterus sphinx (frugivorus bat) and Suncus murinus (house shrew) to Japanese encephalitis virus. Indian J Med Res 79 :8–12.

    • Search Google Scholar
    • Export Citation
  • 16

    Webb NJ, Tidemann CR, 1996. Mobility of Australian flying foxes, Pteropus spp. (Megachiroptera): evidence from genetic variation. Proc R Soc Lond B Biol Sci 263 :497–502.

    • Search Google Scholar
    • Export Citation
  • 17

    Jonsson NN, Johnston SD, Field H, de Jong C, Smith C, 2004. Field anaesthesia of three Australian species of flying fox. Vet Rec 154 :664.

    • Search Google Scholar
    • Export Citation
  • 18

    Beaty BJ, Calisher CH, Shope RE, 1995. Arboviruses. Lennette EH, Lennette DA, Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. Washington, DC: American Public Health Association, 189–212.

  • 19

    Halpin K, Young PL, Field HE, Mackenzie JS, 2000. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 81 :1927–1932.

    • Search Google Scholar
    • Export Citation
  • 20

    Crameri G, Wang LF, Morrissy C, White J, Eaton BT, 2002. A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies. J Virol Methods 99 :41–51.

    • Search Google Scholar
    • Export Citation
  • 21

    Goddard LB, Roth AE, Reisen WK, Scott TW, 2002. Vector competence of California mosquitoes for West Nile virus. Emerg Infect Dis 8 :1385–1391.

    • Search Google Scholar
    • Export Citation
  • 22

    Turell MJ, Kay BH, 1998. Susceptibility of selected strains of Australian mosquitoes (Diptera: Culicidae) to Rift Valley fever virus. J Med Entomol 35 :132–135.

    • Search Google Scholar
    • Export Citation
  • 23

    Pyke AT, Smith IL, van den Hurk AF, Northill JA, Chuan TF, Westacott AJ, Smith GA, 2004. Detection of Australasian flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays. J Virol Methods 117 :161–167.

    • Search Google Scholar
    • Export Citation
  • 24

    Biggerstaff BJ, 2003. PooledInfRate: a Microsoft Excel add-in to compute prevalence estimates from pooled samples. Fort Collins, CO: Centers for Disease Control and Prevention.

  • 25

    Smith IL, Westacott AJ, Smith GA, 2005. Development and implementation of a porcine IgM capture enzyme linked immunosorbent assay for the detection of Japanese encephalitis virus antibodies in sentinel pigs. Arbovirus Res Aus 9 :352–356.

    • Search Google Scholar
    • Export Citation
  • 26

    Pyke AT, Phillips DA, Chuan TF, Smith GA, 2004. Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine. BMC Microbiol 4 :3–10.

    • Search Google Scholar
    • Export Citation
  • 27

    Johnsen DO, Edelman R, Grossman RA, Muangman D, Pomsdhit J, Gould DJ, 1974. Study of Japanese encephalitis virus in Chiangmai Valley, Thailand. V. Animal infections. Am J Epidemiol 100 :57–68.

    • Search Google Scholar
    • Export Citation
  • 28

    Oda K, Igarashi A, Keong CT, Hong CC, Vijayamalar B, Sinniah M, Hassan SS, Tanaka H, 1996. Cross-sectional serosurvey for Japanese encephalitis specific antibody from animal sera in Malaysia 1993. Southeast Asian J Trop Med Public Health 27 :463–470.

    • Search Google Scholar
    • Export Citation
  • 29

    Takashima I, Watanabe T, Ouchi N, Hashimoto N, 1988. Ecologic studies of Japanese encephalitis virus in Hokkaido: interepidemic outbreaks of swine abortion and evidence for the virus to overwinter locally. Am J Trop Med Hyg 38 :420–427.

    • Search Google Scholar
    • Export Citation
  • 30

    Burns KF, 1950. Congenital Japanese B encephalitis infection of swine. Proc Soc Exp Biol Med 75 :621–625.

  • 31

    Lord CC, Rutledge CR, Tabachnick WJ, 2006. Relationships between host viremia and vector susceptibility for arboviruses. J Med Entomol 43 :623–630.

    • Search Google Scholar
    • Export Citation
  • 32

    Gould DJ, Byrne RJ, Hayes DE, 1964. Experimental infection of horses with Japanese encephalitis virus by mosquito bite. Am J Trop Med Hyg 13 :742–746.

    • Search Google Scholar
    • Export Citation
  • 33

    Kay BH, Young PL, Hall RA, Fanning ID, 1985. Experimental infection with Murray Valley encephalitis virus. Pigs, cattle, sheep, dogs, rabbits, macropods and chickens. Aust J Exp Biol Med Sci 63 :109–126.

    • Search Google Scholar
    • Export Citation
  • 34

    Gómez A, Kramer LD, Dupuis AP, Kilpatrick AM, Davis LJ, Jones MJ, Daszak P, Aguirre AA, 2008. Experimental infection of eastern gray squirrels (Sciurus carolinensis) with West Nile virus. Am J Trop Med Hyg 79 :447–451.

    • Search Google Scholar
    • Export Citation
  • 35

    Brown AN, Kent KA, Bennett CJ, Bernard KA, 2007. Tissue tropism and neuroinvasion of West Nile virus do not differ for two mouse strains with different survival rates. Virology 368 :422–430.

    • Search Google Scholar
    • Export Citation
  • 36

    Turell MJ, Tammariello RF, Spielman A, 1995. Nonvascular delivery of St. Louis encephalitis and Venezuelan equine encephalitis viruses by infected mosquitoes (Diptera: Culicidae) feeding on a vertebrate host. J Med Entomol 32 :563–568.

    • Search Google Scholar
    • Export Citation
  • 37

    Turell MJ, Spielman A, 1992. Nonvascular delivery of Rift Valley fever virus by infected mosquitoes. Am J Trop Med Hyg 47 :190–194.

  • 38

    Higgs S, Schneider BS, Vanlandingham DL, Klingler KA, Gould EA, 2005. Nonviremic transmission of West Nile virus. Proc Natl Acad Sci USA 102 :8871–8874.

    • Search Google Scholar
    • Export Citation
  • 39

    McGee CE, Schneider BS, Girard YA, Vanlandingham DL, Higgs S, 2007. Nonviremic transmission of West Nile virus: evaluation of the effects of space, time, and mosquito species. Am J Trop Med Hyg 76 :424–430.

    • Search Google Scholar
    • Export Citation
  • 40

    Markus N, Hall L, 2004. Foraging behaviour of the black flying fox (Pteropus alecto) in the urban landscape of Brisbane, Queensland. Wildl Res 31 :345–355.

    • Search Google Scholar
    • Export Citation
  • 41

    Breed AC, Smith CS, Epstein JH, 2006. Winged wanderers: long distance movements of flying foxes. MacDonald DW, ed. The Encyclopedia of Mammals. Oxford: Oxford University Press, 474–475.

  • 42

    Ritchie SA, Rochester W, 2001. Wind-blown mosquitoes and introduction of Japanese encephalitis into Australia. Emerg Infect Dis 7 :900–903.

    • Search Google Scholar
    • Export Citation
Save