Mosquitoborne viruses, Czech republic, 2002.

Specimens from residents (N = 497) of an area in the Czech Republic affected by the 2002 flood were examined serologically for mosquitoborne viruses. Antibodies were detected against Tahyna (16%), Sindbis (1%), and Batai (0.2%) viruses, but not West Nile virus. An examination of paired serum samples showed 1 Tahyna bunyavirus (California group) infection.

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The 2002 flood in Bohemia struck the Czech Republic I just a few years after the 1997 flood (in Moravia and Silesia). Apart from Prague, extensive rural areas along the Vltava and Labe Rivers were flooded in August 2002. In the Melnik area, which offers favorable habitats for mosquitoes under normal conditions (1), mass mosquito breeding (largely Ochlerotatus sticticus, Oc. cantans, Aedes vexans, and Ae. cinereus) occurred after August 20. This increased mosquito population peaked September 3-9, with a biting frequency of 70 bites per person per minute. The mosquito population declined during the second half of September and disappeared by November.

The Study

To estimate the risk for infections with mosquitobome viruses, we screened the human population of the flooded area (Figure 1) for antibodies against the viruses known to occur in central Europe (2): Tahyna (TAHV), Orthobunyavirus of the California group, Bunraviridae; West Nile (WNV), Flavivirus of the Japanese encephalitis group, Flaviviridae; Sindbis (SINV), AIphavirus, Togaviridae; and Batai (BATV), Orthobunyavirus of the Bunyamwera group, Bunyaviridae.

We subdivided the flooded area into 4 risk zones according to quantities of mosquitoes. Zone A was a forested floodplain along the Labe River between Obristvi-Kly and Lobkovice-Kozly (11 villages), with large quantities of mosquitoes. Zone B was an intermediate area between zones A and C (5 villages, 1 small town), with fewer breeding sites but possibility for mosquito migration from zone A. Zone C was the area along the Vltava and Labe Rivers between Kralupy and Horni Pocaply (25 villages and small towns), with no floodplain forests and few breeding sites for mosquitoes. Zone D was a control zone, with only sporadic occurrences of mosquitoes (mainly in Prague).

Informed written consent and serum samples were obtained from 497 survey participants of various ages from September 6 to September 13, 2002 (3 weeks after the flood culmination and 2 weeks after the mosquito emergence). Paired serum samples were taken from 150 of the survey participants 7 months later, from April 9 to May 15, 2003 (34 in zone A, 43 in zone B, 73 in zone C).

Serologic examination was performed with the hemagglutination-inhibition (HIT) and plaque-reduction neutralization tests (PRNT) in microplates (3-5). The strains used for HIT were TAHV 92, WNV Egl01, BATV 184, and SINV Eg339; a commercial control antigen (Test-Line Ltd., Brno, Czech Republic) of Central European tick-borne encephalitis virus (CEEV) was used. All serum samples were acetone-extracted and tested with sucrose- and acetone-processed antigens by using 8 hemagglutinin units; titers >20 were considered positive. For PRNT, TAHV T16, WNV Egl01, CEEV Hypr, and BAT 184 viral strains were used. The test was conducted on Vero or SPEV (embryonic pig kidney: for CEEV) cells. All serum samples were heat inactivated and screened for antibodies at 1:8; those reducing the number of virus plaques by 90% were considered positive and titrated to estimate dilutions causing plaque--umber reduction by 50% (PRN[T.sub.50]) and 90% (PRN[T.sub.90]). The serum samples reacting with WNV were examined for cross-reactivity with CEEV. PRNT with BATV was used only as a confirmatory test for the serum samples reacting with BATV in HIT.

Against TAHV, 82 (16.5%) of 497 study participants had neutralizing antibodies, and 74 (14.9%) were seropositive in HIT. In PRN[T.sub.50], the titers were 32-2048 (geometric mean titer [GMT] 260), in PRN[T.sub.90] 16-1024 (GMT 119), and in HIT 20-40 to 160 (GMT 40). Figure 2 illustrates the distribution of neutralizing antibody titers. No difference occurred in neutralizing antibody prevalence between sexes, 32 (15.8%) of 202 males and 50 (16.9%) of 295 females ([chi square] - 0.11; p = 0.744). The prevalence rate increased significantly with age (Table 1: [chi square] = 39.809; p <0.001); TAHV antibodies were found infrequently in persons <19 years of age. Neutralizing antibody distribution, with respect to the residence location (Table 2, Figure 1), showed the highest seroprevalence in zone A (28%), lower seroprevalences in zones B and C, and 5% in the control zone D ([chi square] = 14.57; p = 0.002). Significant differences were found between zone D and all other zones, and between zones A and C ([chi square] = 7.243; p = 0.007), but not between zones A and B or B and C; HIT yielded similar results. The seroprevalence in relation to the proximity of study participants' locations to the nearest floodplain forest within zones A, B, and C demonstrated decreasing seroprevalence with increasing distance to the forest ([chi square] = 8.51; p = 0.003) (Table 2).

Against WNV, no specific reactions were found. Although serum samples from 34 (6.8%) study participants reacted in HIT with the WNV at titers 40 to 80, all of them also reacted with CEEV at titers similar or higher ([less than or equal to] 160). CEEV could have occurred in the area, and some study participants may have been vaccinated against tick-borne encephalitis. In PRN[T.sub.90], 6 study participants (1.2%) reacted with WNV but at low titers of 8 to 16; these serum samples also reacted in PRNT with CEEV; thus, the results were considered to be crossreactions as well. Additionally, 42 (8.5%) seroreactors against WNV appeared in the less stringent PRN[T.sub.50], but all titers were low (8-32) and cross-reacted with CEEV.

Against SINV, antibodies were tested with HIT only and detected in specimens from 7 (1.4%) study participants, with low titers of 20 to 40. Of the BATV, specimens from 7 study participants reacted in HIT at a low titer of 20. By confirmatory testing of these serum samples in PRNT, only 1 (0.2%) showed specific antibodies to BATV; the titer was 64 in PRN[T.sub.50] and 32 in PRN[T.sub.90].

Seroconversion ([greater than or equal to] 4-fold rise in titer) was found with TAHV only. After the flood the infection episode occurred in one 55-year-old woman from Obristvi (zone A), as shown by the seroconversion in both HIT (<20/40) and PRN[T.sub.50] (<8/512). Three other study participants seroconverted in 1 test only: a 40-year-old man from Chlumin, zone B (HIT 20/80; PRNT 128/128); a 32-year-old man from Chlumin (HIT

<20/20-40; PRNT 128/64); and an 80-year-old woman from Obristvi (HIT 20/80; PRNT 64/32). These results are less convincing. Upon our request, local general practitioners did not corroborate consistent signs of a disease reported by these 4 study participants from October 2002 to April 2003. In general, clinical symptoms of TAHV infection are milder in adults than in children (7). Seroconversion against mosquitoborne viruses was not detected in any of the 73 study participants in zone C.

Conclusions

On the basis of this serosurvey, recent infections with WNV (in contrast to South Moravia after the 1997 flood [5,6]), SINV, and BATV have not been found in Central Bohemia after the flood. However, activity of another mosquitoborne vials, TAHV, has been found in a natural focus along the Labe River at Neratovice. This focus has so far gone unnoticed (8). Lower frequency of TAHV antibodies has been detected along the lower reaches of the Vltava River. The prevalence of antibodies to TAHV increased with risk-zone ranking (from zone D to the highest risk zone A) and with decreasing distance to floodplain forests, the primary breeding habitat of vector mosquitoes (9-11).

In disease-endemic areas, the proportion of residents with antibodies against California group viruses increase with age (6,12). A similar situation occurred in the Central Bohemian flooded area, where antibodies to TAHV were detected in a low proportion of residents <20 years of age. Nevertheless, TAHV seems to be active in the area. At least 1 seroconversion among 150 residents (attack rate [approximately equal to] 0.67%) against TAHV has been proven. With [approximately equal to]100,000 inhabitants in the risk zones (1992 census), [approximately equal to] 670 (95% confidence interval 20-3,719) persons could have been infected after the flood.

Environmental factors, such as heavy rains followed by a flood, artificial inundation of floodplain forests, or rehabilitation of wetlands that support mosquito-vector populations, could give rise to preconditions for an increased incidence of mosquitoborne infectious diseases, even in temperate climates. Under such circumstances, the optimum strategy is an epidemiologic surveillance that includes monitoring, especially of infection rate of mosquito populations and incidence of mosquitoborne diseases in humans. The surveillance results could then be used in integrated mosquito control.

Table 1. Comparison of the prevalence of neutralizing antibodies to
Tahyna virus by age groups after the floods in central Bohemia in
2002 and southern Moravia in 1997 * ([dagger])

                               CB 2002,     %      SM 1997,     %
           Age (y)                 n     positive     n      positive

0-9                               18       5.6        39       0.0
10-19                             53       0.0        49       8.2
20-29                             74       5.4       128       19.5
30-39                             69       17.4       79       63.3
40-49                             62       11.3       80       62.5
50-59                             86       19.8       90       81.1
60-69                             78       32.1       59       79.7
[greater than or equal to] 70     57       28.1       95       88.4

* CB, central Bohemia; SM, southern Moravia; n, number of residents
examined.

([dagger]) Source (6).

Table 2. Prevalence of neutralizing antibodies to Tahyna virus
after the 2002 flood, Central Bohemia *

                                 n ([dagger])   % positive

Risk zone
A                                     75           28.0
B                                     83           20.5
C                                    279           14.7
D                                     60           5.0

Distance to FPF (km)
<1.0                                  78           28.2
1.0-2.9                               75           21.3
3.0-5.9                               70           17.1
[greater than or equal to] 6.0       214           13.6

* As related to the residence location: risk zones A to D; and
distance to floodplain forest (FPF, within zones A, B, and C only).

([dagger]) n, number of residents examined.

Acknowledgments

We thank the staff members and general practitioners in the Department of Biochemistry of Klaudian Hospital in Mlada Boleslav for collecting blood samples in the area and KlinLab Ltd. for providing blood samples from Prague residents.

The study was supported by the Grant Agency of the Czech Republic (310/03/Z033: 206/03/0726).

Dr. Hubalek is a senior scientist at the Czech Academy of Sciences and an associate professor in microbiology at Masaryk University, Brno, Czech Republic. His research interests include the epidemiology and ecology of arthropodborne microorganisms.

References

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(2.) Hubalek Z, Halouzka J. Arthropod-borne virnses of vertebrates in Europe. Acta Scicntiarnm Naturalium Brno. 1996; 10(4-5): 1-95.

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(6.) Hubalek Z. Halouzka J, Juricova Z, Prikazsky Z, Zakova J, Sebesta O. Surveillance for mosquitoborne viruses in the Breclav area after the 1997 flood. [in Czech]. Epidem Mikrobiol Imunol. 1999;48:91-6.

(7.) Bardos V, Medek M, Kania V, Hubalek Z. Isolation of Tahyna virus from the blood of sick children. Acta Virol. 1975;19:447.

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(9.) Kohnan JM, Malkova D, Nemec A, Smetana A, Hajkova Z, Minar J. The isolation of the Tahyna virus from the mosquito Aeries vexans in southern Moravia. J Hyg Epidem. 1964:8:380-6.

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(12.) Szumlas DE, Apperson CS, Hartig PC, Francy DB. Karabatsos N. Seroepidemiology of La Crosse virus infection in humans in western North Carolina. Am J Trop Med Hyg. 1996;54:332-7.

* Institute of Vertebrate Biology ASCR, Brno, Czech Republic; ([dagger]) Health Institute, Kolin, Czech Republic; and ([double dagger]) Central Bohemia Hygienic Station Prague, Melnik, Czech Republic

Hubalek, * Petr Zeman, ([dagger]) Jiri Halouzka, * Zina Juricova, * Eva Stovickova, ([double dagger]) Helena Balkova, ([dagger]) Silvie Sikutova, * and Ivo Rudolf *

Address for correspondence: Zdenek Hubalek, Laboratory of Medical Zoology UBO AVCR. Klasterni 2, CZ-69142 Valtice, Czech Republic; fax: [phone]: email: [e-mail]

The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

Dr. Schroeder is a risk analyst with the U.S. Department of Agriculture. His general research interests include risk assessment and foodborne illness.