• View in gallery

    Mean (+Std Dev) viremia (log10 plaque forming units (PFU)/mL of blood) for House finches plotted as a function of time in days after inoculation during the initial (V1–V7) and challenge (C1–C6) viremia periods. Minimal threshold for virus detection shown by horizontal arrow was 1.7 log10 PFU/mL.

  • View in gallery

    Mean EIA P/N ratios for House finches tested for EIA antibody using either SLEV (A) or WNV (B) antigen at 6 wks after initial infection (0) and then during each of 6 wks after challenge. Legend shows the order of SLEV (S), WNV (W) or diluent inoculation negative control (C).

  • View in gallery

    Reciprocal of the geometric mean titers for House finches tested for PRNT80 antibody using either SLEV (A) or WNV (B) at 6 wks after initial infection (0) and then at each of 6 wks after challenge. Legend shows the order of SLEV (S), WNV (W), or diluent inoculation negative control (C).

  • 1

    Work TH, 1971. On the Japanese B-West Nile virus complex or an arbovirus problem of six continents. Am J Trop Med Hyg 20 :169–186.

  • 2

    Komar N, 2003. West Nile virus: epidemiology and ecology in North America. Adv Virus Res 61 :185–234.

  • 3

    Hayes CG, 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci 951 :25–37.

  • 4

    Monath TP, 1980. St. Louis Encephalitis. Washington, DC: Am Publ Hlth Assoc.

  • 5

    Lillibridge KM, Parsons R, Randle Y, Travassos da Rosa AP, Guzman H, Siirin M, Wuithiranyagool T, Hailey C, Higgs S, Bala AA, Pascua R, Meyer T, Vanlandingham DL, Tesh RB, 2004. The 2002 introduction of West Nile virus into Harris County, Texas, an area historically endemic for St. Louis encephalitis. Am J Trop Med Hyg 70 :676–681.

    • Search Google Scholar
    • Export Citation
  • 6

    Reisen WK, Lothrop HD, Chiles RE, Madon MB, Cossen C, Woods L, Husted S, Kramer VL, Edman JD, 2004. Invasion of California by West Nile Virus. Emerg Infect Dis 10 :1369–1378.

    • Search Google Scholar
    • Export Citation
  • 7

    Reisen WK, Lothrop HD, Chiles RE, Cusack R, Green E-GN, Fang Y, Kensington M, 2002. Persistence and amplification of St. Louis encephalitis virus in the Coachella Valley of California, 2000–2001. J Med Entomol 39 :793–805.

    • Search Google Scholar
    • Export Citation
  • 8

    Reisen WK, Fang Y, Martinez VM, 2005. Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J Med Entomol 42 :367–375.

    • Search Google Scholar
    • Export Citation
  • 9

    Reisen WK, Milby MM, Presser SB, Hardy JL, 1992. Ecology of mosquitoes and St. Louis encephalitis virus in the Los Angeles Basin of California, 1987–1990. J Med Entomol 29 :582–598.

    • Search Google Scholar
    • Export Citation
  • 10

    Wheeler SS, Reisen WK, Chiles RE, 2004. West Nile infections in free ranging wild birds in the Coachella Valley, Riverside Co., California. Proc Mosq Vector Control Assoc Calif 72 :12–14.

    • Search Google Scholar
    • Export Citation
  • 11

    Wilson J, Hazelrigg JE, Reisen WK, Madon MB, 2004. Invasion of Greater Los Angeles by West Nile virus—2003. Proc Mosq Vector Control Assoc Calif 72 :6–11.

    • Search Google Scholar
    • Export Citation
  • 12

    Reisen WK, Kramer LD, Chiles RE, Green E-GN, Martinez VM, 2001. Encephalitis virus persistence in California birds: preliminary studies with house finches (Carpodacus mexicanus). J Med Entomol 38 :393–399.

    • Search Google Scholar
    • Export Citation
  • 13

    McLean RG, Mullenix J, Kerschner J, Hamm J, 1983. The house sparrow (Passer domesticus) as a sentinel for St. Louis encephalitis. Am J Trop Med Hyg 32 :1120–1129.

    • Search Google Scholar
    • Export Citation
  • 14

    Xiao SY, Guzman H, Zhang H, Tesh RB, Kulasekera VL, Kramer L, Mostashari F, Cherry B, Trock SC, Glaser C, Miller JR, 2001. West Nile virus infection in the golden hamster (Mesocricetus auratus): a model for West Nile encephalitis. Emerg Infect Dis 7 :714–721.

    • Search Google Scholar
    • Export Citation
  • 15

    Tesh RB, Travasos da Rosa AP, Guzman H, Araujo TP, Xiao SY, 2002. Immunization with heterologous flaviviruses protective against fatal West Nile encephalitis. Emerg Infect Dis 8 :245–251.

    • Search Google Scholar
    • Export Citation
  • 16

    Tempelis CH, Reeves WC, Nelson RL, 1976. Species identification of blood meals from Culex tarsalis that had fed on passeriform birds. Am J Trop Med Hyg 25 :744–746.

    • Search Google Scholar
    • Export Citation
  • 17

    Rosen L, Reeves WC, 1954. Studies of avian malaria in vectors and hosts of encephalitis in Kern County, California. Am J Trop Med Hyg 3 :704–708.

    • Search Google Scholar
    • Export Citation
  • 18

    Reeves WC, Asman SM, Hardy JL, Milby MM, Reisen WK, 1990. Epidemiology and Control of Mosquitoborne Arboviruses in California, 1943–1987. Sacramento, CA: Calif. Mosq. Vector Control Assoc.

  • 19

    Herman CM, Reeves WC, McClure HE, French EM, Hammon WM, 1954. Studies on avian malaria in vectors and hosts of encephalitis in Kern County, California. I. Infections in avian hosts. Am J Trop Med Hyg 3 :676–695.

    • Search Google Scholar
    • Export Citation
  • 20

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 21

    Reisen WK, Chiles RE, Martinez VM, Fang Y, Green EN, 2003. Experimental infection of California birds with western equine encephalomyelitis and St. Louis encephalitis viruses. J Med Entomol 40 :968–982.

    • Search Google Scholar
    • Export Citation
  • 22

    Hardy JL, Reeves WC, 1990. Experimental studies on infection in vertebrate hosts. In: Reeves WC, ed. Epidemiology and Control of Mosquitoborne Arboviruses in California, 1943–1987. Sacramento, CA: Calif. Mosq. Vector Control Assoc., 66–127.

  • 23

    Takahashi RM, Reisen WK, Barker CM, 2005. Invasion of Kern County by West Nile virus. Proc Mosq Vector Control Assoc Calif 73 :20–23.

  • 24

    Chiles RE, Reisen WK, 1998. A new enzyme immunoassay to detect antibodies to arboviruses in the blood of wild birds. J Vector Ecol 23 :123–135.

    • Search Google Scholar
    • Export Citation
  • 25

    Reisen WK, Chiles RE, Kramer LD, Martinez VM, Eldridge BF, 2000. Method of infection does not alter the response of chicks and house finches to western equine encephalomyelitis and St. Louis encephalitis viruses. J Med Entomol 37 :250–258.

    • Search Google Scholar
    • Export Citation
  • 26

    Kauffman EB, Jones SA, DuPuis AP, Ngo KA, Bernard KA, Kramer LD, 2003. Virus detection protocols for West Nile virus in vertebrate and mosquito specimens. J Clin Microbiol 41 :3661–3667.

    • Search Google Scholar
    • Export Citation
  • 27

    Steinlein DB, Husted S, Reisen WK, Kramer VL, Chiles RE, Glaser C, Cossen C, Tu E, Gilliam S, Hui LT, Eldridge BF, Boyce K, Yamamoto S, Webb JP, Lothrop HD, Fujioka K, Brisco MJ, Houchin A, Castro M, Hom A, Miles SQ, Rogers C, Cornelius A, McCaughy K, Kohmeier K, Scott TW, 2003. Summary of mosquito-borne encephaltiis virus surveillance in California: 1998–2002. Proc Mosq Vector Control Assoc Calif 71 :17–27.

    • Search Google Scholar
    • Export Citation
  • 28

    Hom A, Houchin A, McCaughey K, Kramer VL, Chiles RE, Reisen WK, Tu E, Glaser C, Cossen C, Baylis E, Eldridge BF, Sun B, Padgett K, Woods L, Marcus L, Hui LT, Castro M, Husted S, 2004. Surveillance for mosquito-borne encephalitis activity and human disease, including West Nile virus in California, 2003. Proc Mosq Vector Control Assoc Calif 72 :48–54.

    • Search Google Scholar
    • Export Citation
  • 29

    Hom A, Marcus L, Kramer VL, Cahoon B, Glaser C, Cossen C, Baylis E, Jean C, Tu E, Eldridge BF, Carney R, Padgett K, Sun B, Reisen WK, Woods L, Husted S, 2005. Surveillance for mosquito-borne encephalitis virus activity and human disease, including West Nile virus, in California, 2004. Proc Mosq Vector Control Assoc Calif 73 :66–77.

    • Search Google Scholar
    • Export Citation
  • 30

    Reisen WK, Lundstrom JO, Scott TW, Eldridge BF, Chiles RE, Cusack R, Martinez VM, Lothrop HD, Gutierrez D, Wright S, Boyce K, Hill BR, 2000. Patterns of avian seroprevalence to western equine encephalomyelitis and St. Louis encephalitis viruses in California, USA. J Med Entomol 37 :507–527.

    • Search Google Scholar
    • Export Citation
  • 31

    Statistical Software NCSS, 2000. Kaysville, UT: NCSS. 1998.

  • 32

    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
  • 33

    Inouye S, Matsuno S, Tsurukubo Y, 1984. “Original antigenic sin” phenomenon in experimental flavivirus infections of guinea pigs: studies by enzyme-linked immunosorbent assay. Microbiol Immunol 28 :569–574.

    • Search Google Scholar
    • Export Citation
  • 34

    Bond JO, Hammon WM, 1970. Epidemiologic studies of possible cross protection between dengue and St. Louis encephalitis arboviruses in Florida. Am J Epidemiol 92 :321–329.

    • Search Google Scholar
    • Export Citation
  • 35

    Tarr GC, Hammon WM, 1974. Cross-protection between group B arboviruses: resistance in mice to Japanese B encephalitis and St. Louis encephalitis viruses induced by Dengue virus immunization. Infect Immun 9 :909–915.

    • Search Google Scholar
    • Export Citation
  • 36

    Goverdhan MK, Kulkarni AB, Gupta AK, Tupe CD, Rodrigues JJ, 1992. Two-way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques. Acta Virol 36 :277–283.

    • Search Google Scholar
    • Export Citation
  • 37

    Kay BH, Fanning ID, Carley JG, 1984. The vector competence of Australian Culex annulirostris with Murray Valley encephalitis and Kunjin viruses. Aust J Exp Biol Med Sci 62 :641–650.

    • Search Google Scholar
    • Export Citation
  • 38

    Hall RA, Broom AK, Smith DW, Mackenzie JS, 2002. The ecology and epidemiology of Kunjin virus. Curr Top Microbiol Immunol 267 :253–269.

 

 

 

 

PREVIOUS INFECTION WITH WEST NILE OR ST. LOUIS ENCEPHALITIS VIRUSES PROVIDES CROSS PROTECTION DURING REINFECTION IN HOUSE FINCHES

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  • 1 Center for Vectorborne Diseases and Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California

House finches are competent hosts for both West Nile and St. Louis encephalitis viruses and frequently become infected during outbreaks. In the current study, House finches were infected initially with either West Nile or St. Louis encephalitis viruses and then challenged 6 weeks post infection with either homologous or heterologous viruses. Although mortality rates were high during initial infection with West Nile virus, prior infection with either virus prevented mortality upon challenge with West Nile virus. Prior infection with West Nile virus provided sterilizing immunity against both viruses, whereas prior infection with St. Louis encephalitis virus prevented viremia from St. Louis encephalitis virus, but only reduced West Nile virus viremia titers. Immunologic responses were measured by enzyme immunoassay and plaque reduction neutralization tests. Heterologous challenge with West Nile virus in birds previously infected with St. Louis encephalitis virus produced the greatest immunologic response, markedly boosting antibody levels against St. Louis encephalitis virus. Our data have broad implications for free-ranging avian serological diagnostics and possibly for the recent disappearance of St. Louis encephalitis virus from California.

St. Louis encephalitis (SLEV) and West Nile (WNV) viruses are members of the Japanese encephalitis serogroup within the genus Flavivirus whose distributions did not overlap prior to the invasion of North America by WNV.13 The introduction of WNV into New York in 1999 was followed rapidly by the dispersal of WNV across North America into areas historically endemic for SLEV, including California, Florida, and Texas.4,5 During the summer of 2003, WNV invaded the southeastern deserts of California,6 an area supporting SLEV transmission,7 thereby allowing us to test the notion that 2 antigenically similar arboviruses may not occupy the same niche at the same time. In southern California, both viruses are transmitted primarily by Culex tarsalis Coquillett in rural landscapes and by Cx. pipiens quinquefasciatus Say in urban settings6,8,9 and presumably are maintained by infections in House finches (Carpodacus mexicanus) and House sparrows (Passer domesticus).2,10,11

Acquisition of protective immunity following infection in the primary avian maintenance hosts could be one of several factors affecting the co-existence of antigenically similar arboviruses. Acquired immunity persists into the following season, because House finches challenged with the same SLEV strain retained protective immunity into the summer after the year of infection, even though neutralizing antibody titers decayed rapidly,12 agreeing with previous results for House sparrows.13 The extent of cross protection from acquired immunity to SLEV against WNV has not been studied in Passeriform birds. Using a hamster model to study WNV infection outcome,14 previous infection or immunization with SLEV and other members of the Japanese encephalitis sero-group protected hamsters against fatal WNV disease but did not prevent viremia during acute infection.15 The reciprocal experiment was not done, although presumably previous WNV infection also would provide cross protection against challenge with SLEV.

The objective of the current experiment was to describe the degree of homologous and heterologous protection afforded by prior WNV or SLEV infection in House finches. We selected House finches for this research because they are abundant throughout California (http://www.mbr-pwrc.usgs.gov/bbs/bbs.html), easy to maintain in captivity, frequently fed upon by Cx. tarsalis,16 a mosquito with which they exchange a number of arboviruses and malaria parasites,1719 and are competent hosts for SLEV and WNV.8,2022 The complexity of avian Flavivirus serology was explored using field samples from 2 study areas in California with active WNV transmission, but with differing recent histories of SLEV transmission.

MATERIALS AND METHODS

Virus strains.

We used the NY strain of WNV isolated from a Flamingo that died in the Bronx Zoo (strain 35211 AAF 9/23/99) and that previously was passaged twice in Vero cells. The Kern217 strain of SLEV isolated from Cx. tarsalis collected in Bakersfield in 1989 also had been passaged twice in Vero cells prior to use. Both virus strains have been used extensively in host competence studies in our laboratory.8,21

Avian infection.

House finches were collected from Vineyards near Bakersfield during the summer of 2003, before the introduction of WNV into Kern County, California.23 Birds were banded, bled to determine antibody status, and maintained for 1–2 weeks to observe general health and adaptation to confinement. Sera taken prior to infection were tested for antibodies against western equine encephalomyelitis virus (Togaviridae, Alphavirus, WEEV) and SLEV using an enzyme immunoassay (EIA).24 Birds were fed mixed bird seed and housed in mosquito-proofed and air-conditioned infection units.

Birds were inoculated subcutaneously with 3–4 log10 PFU of each virus in the cervical region. Previous studies have shown that comparable titers of SLEV delivered by syringe or infectious mosquito bite produced similar viremia and antibody responses in House finches.25 Birds were bled by jugular venipuncture (0.1 mL blood taken by 28 g syringe and expelled into 0.4 mL virus diluent) daily for 6–7 days post inoculation (dpi) to monitor viremia and then weekly for 4–6 weeks to monitor antibody (0.1 mL blood into 0.9 mL saline). Virus diluent contained buffered saline, 20% fetal bovine sera and antibiotics. Initially, 16 birds were inoculated with each virus and 16 birds were inoculated with virus diluent and bled concurrently as negative controls. Because of elevated mortality among birds infected with WNV, 20 additional birds were infected with WNV and 6 of these that survived acute infection were added to the previously WNV-infected group to measure the effects of heterologous challenge with SLEV. During challenge, 4 previous control birds each were infected for the first time with WNV or SLEV as positive controls, whereas 8 birds were inoculated with diluent to measure any possible nonspecific reactivity to fetal bovine sera included in our virus diluent and to monitor mortality related to handling, blood sampling, and maintenance.

Assays.

Viremia titers were measured by standard plaque assay on Vero cell culture.26 Antibody response was measured by an indirect EIA and by a plaque reduction neutralization assay (PRNT) using virus grown on Vero cell culture.24 To be considered positive, EIA antigen positive over antigen negative well optical density (P/N) ratios had to be > 2.0 and sera had to neutralize > 80% of virus at a dilution of 1:20. For statistical analyses, we used the loge transformed reciprocal of the PRNT80 end point titers for each virus; means presented graphically were the back transformed reciprocal of geometric mean titers.

Field data.

To illustrate that cross reactivity may be a problem when testing sera from field-collected free-ranging birds, we compared results from our field sampling program in Los Angeles County without a history of recent SLEV activity to Coachella Valley, Riverside County, with a recent history of SLEV activity.2729 Birds were collected as part of our long-term studies on free-ranging avian seroprevalence at these same areas.30 Recent surveillance reporting the testing of mosquitoes and sentinel chickens in California were summarized from weekly Arbovirus Surveillance Bulletins.

RESULTS

All birds used in these experiments were negative for antibody against WEEV and SLEV when tested prior to infection. All control birds survived our maintenance and bleeding protocols and remained negative for WNV and SLEV and antibodies when tested by plaque assay, EIA, and PRNT during initial and challenge infections.

Mortality post infection was detected only in birds infected for the first time with WNV (Table 1). Ten of 16 birds died during the initial WNV infection and 3 of 4 positive controls died during challenge on 6–7 dpi. In addition, 12 of 20 birds (60%) in the second WNV infection died on 6–8 dpi (data not shown). None of the birds infected with SLEV or inoculated with diluent died. Control birds were housed in separate cages within the same building and bled concurrently with WNV-inoculated birds. Prior infection with either WNV or SLEV prevented mortality in all birds after homologous or heterologous challenge.

Viremia profiles for birds initially infected and challenged were measured daily up to 7 dpi (Figure 1). Mean viremia for birds infected with WNV peaked on 3 dpi at 7.3 and 8.0 log10 PFU/mL during the initial infection and in the positive control group monitored during challenge, respectively. In marked contrast, birds initially infected with SLEV produced viremias that peaked on 3 and 2 dpi at 3.0 and 2.8 log10 PFU/mL for initial infection and positive control groups, respectively. Birds surviving previous infection with WNV produced sterilizing immunity against both WNV and SLEV at challenge 6 weeks post infection (i.e., virus was not detected in birds within WNV:WNV or WNV:SLEV groups). In contrast, although initial infection with SLEV produced protective immunity upon homologous challenge with SLEV, birds challenged with WNV exhibited a viremia that peaked on 3 dpi at a mean of 4.6 (range, 2.7–6.4) log10 PFU/mL.

The immune response to initial and challenge infections was monitored for both SLEV and WNV using EIA and PRNT (Figures 2 and 3). Antibody at 6 weeks after initial infection in challenged birds and before initial infection for positive and negative controls was compared with antibody levels during 6 weeks after infection using a repeated measures ANOVA31 with virus treatment as the main effect. All serological test results except for the negative controls showed a significant increase over time. When sera were tested by EIA using SLEV antigen, mean positive over negative well ratios varied significantly among virus treatments (F = 11.6, df = 6, 29, P < 0.001), bleed dates (F = 7.4, df = 6, 36; P < 0.001), and virus by date interaction (F = 3.3, df = 36,167; P < 0.001). As shown in Figure 2A, ratios increased most rapidly for the SLEV:SLEV group, followed by the WNV:SLEV group. Lowest ratios were observed for initial infections with WNV. A similar pattern was seen for neutralizing antibody detected by PRNT using SLEV; i.e., virus treatment (F = 21.9; df = 6, 29; P < 0.001), weeks post challenge (F = 7.1; df = 6, 36; P < 0.001), and interaction (F = 5.4; df = 36, 156; P < 0.001) terms were all highly significant in the ANOVA. Examination of Figure 3A revealed that most variation was caused by the pronounced antibody response by the SLEV:WNV group, which had a backtransformed geometric mean titer peaking on week 2 post challenge at 1:3,600. Interestingly, the heterologous challenge created a response that was greater than the homologous challenge response that peaked at 1:250 on week 4.

The mean response to WNV antigen or virus was somewhat different. When sera were tested by EIA using WNV antigen, mean positive over negative well ratios varied significantly among virus treatments (F = 43.1, df = 5, 22, P < 0.001), bleed dates (F = 10.1, df = 6, 30; P < 0.001), and virus by date interaction (F = 2.2, df = 30,116; P < 0.001). Highest values were observed for the WNV:SLEV heterologous challenge group, followed by the WNV:WNV homologous challenge group (Fig. 2B). In general, P/N ratio values for WNV antigen were lower than observed for SLEV antigen (Fig. 2A), and the SLEV-positive control group remained negative throughout; i.e., P/N < 2.0. Even the SLEV:SLEV group failed to elicit a cross reactive response > 3.0. All 3 factors in the ANOVA were again highly significant when sera was tested by PRNT using WNV; i.e., virus treatment (F = 65.1; df = 6, 29; P < 0.001), weeks post challenge (F = 30.9; df = 6, 36; P < 0.001), and interaction (F = 6.3; df = 36, 161; P < 0.001) were all highly significant. Again the heterologous SLEV:WNV challenge elicited the highest PRNT titer peaking at 1:3,041 at week 2 post challenge, similar to SLEV PRNT results (Fig. 3B). Similar to the EIA results, the homologous WNV:WNV challenge produced the next highest titers.

The immunologic responses of individual birds varied markedly within treatment groups, even though all birds were infected initially as indicated by positive viremia and/or EIA test results (Table 2). PRNT end point titrations on week 6 (A6) after initial infection with SLEV gave variable results; for example, 5 birds had SLEV antibody titers < 4× WNV titers. The reciprocal was less variable, with only bird 2480 having equivocal titers. After homologous challenge, all birds except 2627 exhibited titers > 4× the competing virus. Differences between end point titers were markedly greater for the WNV:WNV than the SLEV:SLEV group. Heterologous challenge produced different results depending on the order of virus infection. On week C2 after heterologous challenge in the SLEV:WNV group, birds 2652 and 2656 had titers for SLEV > 4× WNV, birds 2649, 2651, and 2656 had titers for WNV > 4× SLEV, and birds 2650, 2653, and 2654 had titers for WNV = SLEV. By week C6 titers in all birds, except 2653, decreased markedly, and all birds except 2652 and 2655 had WNV titers > SLEV titers. When the order of infection was reversed, all birds except 2484 on week C2 had WNV titers > SLEV.

These diagnostic problems manifested themselves during our field studies in California during 2004 (Table 3). In the Coachella Valley where both SLEV and WNV were active during the previous summer, we could only identify 69% of EIA positive sera as either WNV or SLEV; significantly less (χ2= 60.2, df = 1, P < 0.001) than the 91% confirmed from Los Angeles where only WNV was active during both years. In Coachella Valley a significantly larger percentage of the EIA positive sera (χ2 = 66.7, df = 2, P < 0.001) were negative by PRNT80 [13%] or the results were equivocal with low titered end points [18%] than observed in Los Angeles. These latter results most likely were due to old infections, because our studies have shown that PRNT titers decay faster than EIA P/N ratios.12,21

DISCUSSION

West Nile virus virulence in House finches exceeded SLEV as indicated by elevated mortality rates and viremia titers. Previous infection with either SLEV or WNV protected House finches from mortality during subsequent challenge, similar to results observed recently using a hamster laboratory model.15 Sterilizing immunity during homologous and heterologous challenge with SLEV prevented detection of a viremia (i.e., viremia remained < 1.7 log10 PFU/mL, our threshold of detection using Vero cell plaque assay). In contrast, heterologous challenge with WNV in birds previously infected with SLEV resulted in peak viremias that ranged from 2.7–6.4 log10 PFU/mL. Viremias > 5 log10 PFU/mL were considered sufficiently elevated to infect susceptible populations of California Culex mosquitoes.8,32 Interestingly, infection with SLEV following recovery from WNV infection elicited a consistent and significant rise in WNV PRNT, but not SLEV PRNT titers, perhaps because protective immunity prevented the immunologic response associated with a second viremia episode. This pattern is consistent with the concept of “original antigenic sin” previously recognized in mammalian hosts with sequential Flavivirus infections.33 In contrast, infection with WNV following recovery from SLEV produced a very high antibody titers and a non-specific response that was highly variable among individual birds within this treatment group. Differences here were attributed to differential virulence associated with SLEV and WNV infection.

Our serology data pointed out problems in monitoring free-ranging bird seroprevalence when SLEV and WNV are transmitted concurrently and sympatrically. Our field studies screen bird sera using an EIA and confirm possible positives with a P/N ratio > 2 using a PRNT against WNV or SLEV. For confirmation, the PRNT titer against the target virus must be ≥4× the titer against the competing virus. In the current experiment 6 weeks after initial infection we would only have been able to confirm 8 of 14 birds infected with SLEV; 6 birds had PRNT80 titers for SLEV < 4× for WNV. More WNV infections produced a specific antibody response and 11 of 12 birds had PRNT80 titers against WNV > 4× against SLEV. Determination of the original or second infecting virus was virtually impossible after heterologous challenge, because some birds boosted antibody levels against the original virus, some produced equivocal cross-reacting antibody, and others produced antibody titers highest against the second infecting virus. Even reinfection with SLEV produced cross-reacting antibodies by week 6 post challenge that gave equivocal end point titers for WNV and SLEV. The impact of multiple sympatric Flavivirus transmission on avian diagnostics was demonstrated by comparing serological test results on birds collected in Coachella Valley where both WNV was active in 2003 and SLEV active since 2000 to Los Angeles where only WNV was active during 2003. We were able to confirm and identify only 69% of the EIA-positive sera from Coachella Valley, but 91% of sera from Los Angeles. We were able to confirm 87% of the EIA positives from Coachella Valley, but the infecting virus could not be ascertained in 18% of confirmed positives.

Although not well studied in birds, incomplete cross protection with members of the Japanese encephalitis serogroup has been observed in humans34 and other mammals15,35,36 leading to the notion that 2 members of this serogroup cannot coexist.1 Australia seems to be a notable exception with both Kunjin and Murray Valley encephalitis endemic and transmitted principally by the same vector mosquito.37,38 The ongoing WNV epidemic in California has been associated with the disappearance of SLEV, but not the Alphavirus WEEV, which was detected throughout southern California during 2005. We have been tracking the occurrence of WEEV, SLEV, and WNV in Coachella Valley in SE California for the past 18 years.7 SLEV was detected during 15 of the 18 years, was present when WNV invaded southern California in 2003,6 but was not detected during 2004 and 2005, despite the testing of 1,599 and 3,021 pools of mostly Culex mosquitoes, respectively. Similarly, SLEV was not detected throughout California during 2004 or 2005 despite the testing of 14,698 and 20,123 mosquito pools and 32,616 and 32,768 sentinel chicken sera, respectively. Continued monitoring during the subsidence of the WNV epidemic may delineate the intensity of WNV activity and avian ‘herd immunity’ required to prevent the re-emergence of SLEV.

Table 1

Mortality (dead) in house finches following infection with either WNV or SLEV

GroupnDead (%)
WNV, West Nile virus; SLEV, St. Louis encephalitis virus.
Neg, negative controls inoculated with virus diluent.
Pos, positive controls infected for the first time.
In challenge groups, viruses are listed by first:second inoculations.
Initial infection
    WNV1663
    SLEV160
    Neg control160
Challenge
    WNV:WNV40
    WNV:SLEV80
    Pos control WNV475
    Neg control120
    SLEV:SLEV80
    SLEV:WNV80
    Pos control SLEV40
    Neg control80
Table 2

Variable PRNT responses of individual birds to homologous and heterologous challenge

A6C2C6
VirusBandWNVSLEVWNVSLEVWNVSLEV
WNV, West Nile virus; SLEV, St. Louis encephalitis virus.
Data shown are the inverse of the PRNT80 end point titers using either WNV or SLEV on wk 6 after initial infection (A6) and at wks 2 (C2) and 6 (C6) after heterologous or homologous challenge.
SLEV:WNV2649< 20402,5601,280640320
2650408020,48020,480640320
2651< 20401,280640640160
2652< 20402,56020,480320320
265320401,2801,2802,560320
265440402,5602,5601,280320
2655< 20202,56020,480320640
2656< 20< 205,1201,2801,280160
SLEV:SLEV2623203204064020640
2624806408064040320
2625201602080< 2040
2626< 20< 204080< 2080
26278016080808080
262820802016020160
WNV:SLEV26021604010,2406402,56040
2604160403204064040
246780< 201,280801,28080
24778040804032040
247880403202032040
2480202080< 2032020
248480< 20808032080
248780< 20320< 201,28020
WNV:WNV26138020640401,28040
2614160201,280201,28040
2615160405,120802,56040
2616160402,650202,56020
Table 3

Confirmation of positive EIA results using PRNT80 end point titrations against WNV and SLEV for avian sera collected from wild-caught birds in Los Angeles and Coachella Valley during 2004

Los AngelesCoachellaTotal
EIA, enzyme immunoassay; WNV, West Nile virus; SLEV, St. Louis encephalitis virus.
WNV ~SLEV if there was < 4× difference in antibody titers; negative samples had antibody titers against both WNV and SLEV < 1:20.
Tested by PRNT402301703
WNV369196565
SLEV01212
WNV ~SLEV285381
Negative54045
Figure 1.
Figure 1.

Mean (+Std Dev) viremia (log10 plaque forming units (PFU)/mL of blood) for House finches plotted as a function of time in days after inoculation during the initial (V1–V7) and challenge (C1–C6) viremia periods. Minimal threshold for virus detection shown by horizontal arrow was 1.7 log10 PFU/mL.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 3; 10.4269/ajtmh.2006.75.480

Figure 2.
Figure 2.

Mean EIA P/N ratios for House finches tested for EIA antibody using either SLEV (A) or WNV (B) antigen at 6 wks after initial infection (0) and then during each of 6 wks after challenge. Legend shows the order of SLEV (S), WNV (W) or diluent inoculation negative control (C).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 3; 10.4269/ajtmh.2006.75.480

Figure 3.
Figure 3.

Reciprocal of the geometric mean titers for House finches tested for PRNT80 antibody using either SLEV (A) or WNV (B) at 6 wks after initial infection (0) and then at each of 6 wks after challenge. Legend shows the order of SLEV (S), WNV (W), or diluent inoculation negative control (C).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 3; 10.4269/ajtmh.2006.75.480

*

Address correspondence to William K. Reisen, Center for Vectorborne Diseases, Old Davis Rd., Davis, CA 95616. E-mail: arbo123@pacbell.net

The collection and infection of House finches was done under Protocol 11184 approved by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis, California Resident Scientific Collection Permit No. 801049-02 from the State of California Department of Fish and Game, and Federal Fish and Wildlife Permit No. MB082812-0 from the Department of the Interior. Use of arboviruses was approved under Biological Use Authorization #0554 by Environmental Health and Safety of the University of California, Davis, and USDA Permit #47901.

Acknowledgments: The authors thank V. Martinez, B. Carroll, L. Kosareff, and S. Halam, Arbovirus Field Station, for help with the collection and maintenance of the birds, and Sandra Garcia and Siranoosh Ashtari, Arbovirus Laboratory, for help with the EIA and PRNT tests. Sera from free-ranging birds were collected by Sarah Wheeler in Coachella Valley and by Jennifer Wilson in Los Angeles County. This research was funded by Research Grant R01-A155607 from the National Institutes of Allergy and Infectious Diseases, NIH, grants from the University of California Mosquito Research Program, and supplemental funds from the Centers for Disease Control and Prevention and the Coachella Valley MVCD. Logistical support was provided by the Coachella Valley, Greater Los Angeles County and Kern MVCDs. AC Brault, Center for Vectorborne Diseases, critically read the manuscript.

REFERENCES

  • 1

    Work TH, 1971. On the Japanese B-West Nile virus complex or an arbovirus problem of six continents. Am J Trop Med Hyg 20 :169–186.

  • 2

    Komar N, 2003. West Nile virus: epidemiology and ecology in North America. Adv Virus Res 61 :185–234.

  • 3

    Hayes CG, 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Ann N Y Acad Sci 951 :25–37.

  • 4

    Monath TP, 1980. St. Louis Encephalitis. Washington, DC: Am Publ Hlth Assoc.

  • 5

    Lillibridge KM, Parsons R, Randle Y, Travassos da Rosa AP, Guzman H, Siirin M, Wuithiranyagool T, Hailey C, Higgs S, Bala AA, Pascua R, Meyer T, Vanlandingham DL, Tesh RB, 2004. The 2002 introduction of West Nile virus into Harris County, Texas, an area historically endemic for St. Louis encephalitis. Am J Trop Med Hyg 70 :676–681.

    • Search Google Scholar
    • Export Citation
  • 6

    Reisen WK, Lothrop HD, Chiles RE, Madon MB, Cossen C, Woods L, Husted S, Kramer VL, Edman JD, 2004. Invasion of California by West Nile Virus. Emerg Infect Dis 10 :1369–1378.

    • Search Google Scholar
    • Export Citation
  • 7

    Reisen WK, Lothrop HD, Chiles RE, Cusack R, Green E-GN, Fang Y, Kensington M, 2002. Persistence and amplification of St. Louis encephalitis virus in the Coachella Valley of California, 2000–2001. J Med Entomol 39 :793–805.

    • Search Google Scholar
    • Export Citation
  • 8

    Reisen WK, Fang Y, Martinez VM, 2005. Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J Med Entomol 42 :367–375.

    • Search Google Scholar
    • Export Citation
  • 9

    Reisen WK, Milby MM, Presser SB, Hardy JL, 1992. Ecology of mosquitoes and St. Louis encephalitis virus in the Los Angeles Basin of California, 1987–1990. J Med Entomol 29 :582–598.

    • Search Google Scholar
    • Export Citation
  • 10

    Wheeler SS, Reisen WK, Chiles RE, 2004. West Nile infections in free ranging wild birds in the Coachella Valley, Riverside Co., California. Proc Mosq Vector Control Assoc Calif 72 :12–14.

    • Search Google Scholar
    • Export Citation
  • 11

    Wilson J, Hazelrigg JE, Reisen WK, Madon MB, 2004. Invasion of Greater Los Angeles by West Nile virus—2003. Proc Mosq Vector Control Assoc Calif 72 :6–11.

    • Search Google Scholar
    • Export Citation
  • 12

    Reisen WK, Kramer LD, Chiles RE, Green E-GN, Martinez VM, 2001. Encephalitis virus persistence in California birds: preliminary studies with house finches (Carpodacus mexicanus). J Med Entomol 38 :393–399.

    • Search Google Scholar
    • Export Citation
  • 13

    McLean RG, Mullenix J, Kerschner J, Hamm J, 1983. The house sparrow (Passer domesticus) as a sentinel for St. Louis encephalitis. Am J Trop Med Hyg 32 :1120–1129.

    • Search Google Scholar
    • Export Citation
  • 14

    Xiao SY, Guzman H, Zhang H, Tesh RB, Kulasekera VL, Kramer L, Mostashari F, Cherry B, Trock SC, Glaser C, Miller JR, 2001. West Nile virus infection in the golden hamster (Mesocricetus auratus): a model for West Nile encephalitis. Emerg Infect Dis 7 :714–721.

    • Search Google Scholar
    • Export Citation
  • 15

    Tesh RB, Travasos da Rosa AP, Guzman H, Araujo TP, Xiao SY, 2002. Immunization with heterologous flaviviruses protective against fatal West Nile encephalitis. Emerg Infect Dis 8 :245–251.

    • Search Google Scholar
    • Export Citation
  • 16

    Tempelis CH, Reeves WC, Nelson RL, 1976. Species identification of blood meals from Culex tarsalis that had fed on passeriform birds. Am J Trop Med Hyg 25 :744–746.

    • Search Google Scholar
    • Export Citation
  • 17

    Rosen L, Reeves WC, 1954. Studies of avian malaria in vectors and hosts of encephalitis in Kern County, California. Am J Trop Med Hyg 3 :704–708.

    • Search Google Scholar
    • Export Citation
  • 18

    Reeves WC, Asman SM, Hardy JL, Milby MM, Reisen WK, 1990. Epidemiology and Control of Mosquitoborne Arboviruses in California, 1943–1987. Sacramento, CA: Calif. Mosq. Vector Control Assoc.

  • 19

    Herman CM, Reeves WC, McClure HE, French EM, Hammon WM, 1954. Studies on avian malaria in vectors and hosts of encephalitis in Kern County, California. I. Infections in avian hosts. Am J Trop Med Hyg 3 :676–695.

    • Search Google Scholar
    • Export Citation
  • 20

    Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M, 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 9 :311–322.

    • Search Google Scholar
    • Export Citation
  • 21

    Reisen WK, Chiles RE, Martinez VM, Fang Y, Green EN, 2003. Experimental infection of California birds with western equine encephalomyelitis and St. Louis encephalitis viruses. J Med Entomol 40 :968–982.

    • Search Google Scholar
    • Export Citation
  • 22

    Hardy JL, Reeves WC, 1990. Experimental studies on infection in vertebrate hosts. In: Reeves WC, ed. Epidemiology and Control of Mosquitoborne Arboviruses in California, 1943–1987. Sacramento, CA: Calif. Mosq. Vector Control Assoc., 66–127.

  • 23

    Takahashi RM, Reisen WK, Barker CM, 2005. Invasion of Kern County by West Nile virus. Proc Mosq Vector Control Assoc Calif 73 :20–23.

  • 24

    Chiles RE, Reisen WK, 1998. A new enzyme immunoassay to detect antibodies to arboviruses in the blood of wild birds. J Vector Ecol 23 :123–135.

    • Search Google Scholar
    • Export Citation
  • 25

    Reisen WK, Chiles RE, Kramer LD, Martinez VM, Eldridge BF, 2000. Method of infection does not alter the response of chicks and house finches to western equine encephalomyelitis and St. Louis encephalitis viruses. J Med Entomol 37 :250–258.

    • Search Google Scholar
    • Export Citation
  • 26

    Kauffman EB, Jones SA, DuPuis AP, Ngo KA, Bernard KA, Kramer LD, 2003. Virus detection protocols for West Nile virus in vertebrate and mosquito specimens. J Clin Microbiol 41 :3661–3667.

    • Search Google Scholar
    • Export Citation
  • 27

    Steinlein DB, Husted S, Reisen WK, Kramer VL, Chiles RE, Glaser C, Cossen C, Tu E, Gilliam S, Hui LT, Eldridge BF, Boyce K, Yamamoto S, Webb JP, Lothrop HD, Fujioka K, Brisco MJ, Houchin A, Castro M, Hom A, Miles SQ, Rogers C, Cornelius A, McCaughy K, Kohmeier K, Scott TW, 2003. Summary of mosquito-borne encephaltiis virus surveillance in California: 1998–2002. Proc Mosq Vector Control Assoc Calif 71 :17–27.

    • Search Google Scholar
    • Export Citation
  • 28

    Hom A, Houchin A, McCaughey K, Kramer VL, Chiles RE, Reisen WK, Tu E, Glaser C, Cossen C, Baylis E, Eldridge BF, Sun B, Padgett K, Woods L, Marcus L, Hui LT, Castro M, Husted S, 2004. Surveillance for mosquito-borne encephalitis activity and human disease, including West Nile virus in California, 2003. Proc Mosq Vector Control Assoc Calif 72 :48–54.

    • Search Google Scholar
    • Export Citation
  • 29

    Hom A, Marcus L, Kramer VL, Cahoon B, Glaser C, Cossen C, Baylis E, Jean C, Tu E, Eldridge BF, Carney R, Padgett K, Sun B, Reisen WK, Woods L, Husted S, 2005. Surveillance for mosquito-borne encephalitis virus activity and human disease, including West Nile virus, in California, 2004. Proc Mosq Vector Control Assoc Calif 73 :66–77.

    • Search Google Scholar
    • Export Citation
  • 30

    Reisen WK, Lundstrom JO, Scott TW, Eldridge BF, Chiles RE, Cusack R, Martinez VM, Lothrop HD, Gutierrez D, Wright S, Boyce K, Hill BR, 2000. Patterns of avian seroprevalence to western equine encephalomyelitis and St. Louis encephalitis viruses in California, USA. J Med Entomol 37 :507–527.

    • Search Google Scholar
    • Export Citation
  • 31

    Statistical Software NCSS, 2000. Kaysville, UT: NCSS. 1998.

  • 32

    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
  • 33

    Inouye S, Matsuno S, Tsurukubo Y, 1984. “Original antigenic sin” phenomenon in experimental flavivirus infections of guinea pigs: studies by enzyme-linked immunosorbent assay. Microbiol Immunol 28 :569–574.

    • Search Google Scholar
    • Export Citation
  • 34

    Bond JO, Hammon WM, 1970. Epidemiologic studies of possible cross protection between dengue and St. Louis encephalitis arboviruses in Florida. Am J Epidemiol 92 :321–329.

    • Search Google Scholar
    • Export Citation
  • 35

    Tarr GC, Hammon WM, 1974. Cross-protection between group B arboviruses: resistance in mice to Japanese B encephalitis and St. Louis encephalitis viruses induced by Dengue virus immunization. Infect Immun 9 :909–915.

    • Search Google Scholar
    • Export Citation
  • 36

    Goverdhan MK, Kulkarni AB, Gupta AK, Tupe CD, Rodrigues JJ, 1992. Two-way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques. Acta Virol 36 :277–283.

    • Search Google Scholar
    • Export Citation
  • 37

    Kay BH, Fanning ID, Carley JG, 1984. The vector competence of Australian Culex annulirostris with Murray Valley encephalitis and Kunjin viruses. Aust J Exp Biol Med Sci 62 :641–650.

    • Search Google Scholar
    • Export Citation
  • 38

    Hall RA, Broom AK, Smith DW, Mackenzie JS, 2002. The ecology and epidemiology of Kunjin virus. Curr Top Microbiol Immunol 267 :253–269.

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