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1 Department of Virology, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland;
2 Department of Virology, HUSLAB, Hospital District of Helsinki and Uusimaa, FIN-00029, Finland;
3 Department of Clinical Microbiology, Dokuz Eylul University Hospital, 35340 Izmir, Turkey;
4 Centre for Comparative Infectious Diseases, The University of Liverpool, Leahurst CH64 7TE, United Kingdom;
5 Finnish Forest Research Institute, FIN-01301, Vantaa, Finland;
6 Department of Zoology Nig de, Turkey Nig de University, 51200 Nig de, Turkey
8 Corresponding author (email: juha.laakkonen{at}helsinki.fi)
ABSTRACT:
Wild rodents (n = 330) were trapped around the villages of Altindere and Coandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Ç amlihemin, Rize Province), and Bozdag (Ödemi, I zmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirussero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.
Key words: Arenaviruses, hantaviruses, poxviruses, rodent-borne viruses, rodents, seroprevalence.
Although many rodent-borne viruses are known to occur in Europe and adjoining areas (Kallio-Kokko et al., 2005), little is known about the diversity, distribution, or host range of rodent-borne viruses in Turkey (Kavukcu et al., 1997). Hantavirus-specific IgG antibodies, as detected by immunofluorescence assay (IFA), have been reported in 4.3% of patients from Izmir, Turkey, with acute and chronic renal failure (Kavukcu et al., 1997). Human cases of hemorrhagic fever also have occurred in the Giresun and Trabzon areas. As part of our ongoing project on viral zoonoses in Europe and adjoining areas, wild rodents trapped from several sites in northeastern and western Turkey were tested for arenavirus, hanta-virus, and poxvirus antibodies. Human infections with hantaviruses and arena-viruses are incidental to the natural cycle of the viruses; transmission to humans usually occurs by inhalation of aerosolized rodent excreta. Cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) transmission generally is associated with skin abrasions. Wild rodents rarely transmit poxviruses directly to humans, and transmission usually occurs through contact with infected free-roaming cats, or other pets.
Arenaviruses (family Arenaviridae, genus Arenavirus) and hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped negative-stranded RNA viruses. These viruses are usually associated with specific rodent reservoirs. The only arenavirus reported from Europe is lymphocytic choriomeningitis virus (LCMV), which is associated with the common house mouse, Mus musculus (Armstrong and Sweet, 1963). Five hantaviruses, Puumala virus (PUUV), Dobrava-Belgrade virus (DOBV), Saaremaa virus (SAAV), Seoul virus, and Tula virus (TULV), are known from rodents in Europe; with the exception of TULV, these viruses may cause hemorrhagic fever with renal syndrome in humans (Kallio-Kokko et al., 2005). Orthopoxviruses (family Poxviridae, genus Orthopoxvirus) are large DNA viruses found in a variety of vertebrates (Kallio-Kokko et al., 2005); rodents are the reservoir hosts of CPXV (Chantrey et al., 1999). The objective of this study was to serologically test wild rodents for antibodies against these rodent-borne virus antibodies to provide preliminary information on the diversity, distribution, and prevalence of these viruses in Turkey. Efforts also were made to confirm the presence of these viruses by using polymerase chain reaction (PCR).
Rodents (n = 330; Table 1
) were trapped with snap-traps (Finnish type) in several locations around the villages of Altindere and Coandere (40°45'N, 39°37'E; 848 m above sea level, Maçka, Trabzon Province), Ayder (41°00'N, 41°03'E; 932 m) and Ortan and Yolkiyi (41°01'N, 40°59'E; 443 m, Ç amlihemin, Rize Province) in northeastern Turkey; and in Bozdag (38°21'N, 28°05'E; 1,438 m, Ödemi, I zmir Province) in western Turkey during April 2004. Each site was trapped for 2 to 3 days. Traps (n = 300) were placed in areas with signs of rodent activity and checked every 8 hr. Necropsies were done in a field laboratory, and samples of heart, lung, kidney, and spleen were collected; these samples were stored in sterile vials on dry ice in the field. In the laboratory, samples were stored at –70 C or in RNAlater® (Ambion Inc., Huntingdon, UK) at 4 C. Several sets of instruments were used for dissection, and they were sterilized immediately after use. Protective masks and gloves were used during sample collection. Samples were transported to the Department of Virology (Haartman Institute, University of Helsinki, Helsinki, Finland), where all testing was done.
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For hantaviruses, RNA isolation and reverse transcription (RT)-PCR were done as described previously (Plyusnin et al., 1995). Briefly, total RNA was isolated from samples of lung tissue from four seropositive Microtus by using TriPure reagent (Roche Diagnostics, Mannheim, Germany). Attempts to synthesize full-length S segment cDNAs were made using primer 5'-TAGTAGTAGAC-3' and random hexamers. PCR was done with a single primer 5'-TTCTGCAGTAGTAG-TAGACTCCTTGAAAAG-3'. Because no PCR products corresponding to the full-length S segment (~ 1,800 base pairs) were observed using this technique, a nested PCR with primers based on TULV S segment sequence also was performed (Plyusnin et al., 1995).
For poxviruses, lung samples stored in RNAlater® were extracted for DNA as described previously (Bown et al., 2003). DNA was screened using a nested PCR targeting the fusion protein gene (adapted from Chantrey et al., 1999). DNA was screened for orthopoxvirus-specific DNA, by using a nested PCR targeting the fusion protein gene (Chantrey et al., 1999). Cycle conditions were adapted from Chantrey et al. (1999) and consisted of 94 C for 6 min (one cycle), 94 C for 30 sec, 50 C for 30 sec, and 72 C for 1 min (35 cycles), and 72 C for 10 min (one cycle).
Nucleic acid extraction from LCMV-IFA-positive animals (kidney and spleen tissue samples) and RT-PCR were done as described above for hantaviruses. DNA amplification was done using forward primers Lassa 2 (5'-GCC ACA CGT GGC AAA ATT GT) and Lassa 3 (5'-CCCACAGGTGGCAAAATTGT) and reverse primer Lassa 1 (5'-GCA CCG GGG ATC CTA GGC AT). The PCRs were performed in a total volume of 50 µl of AmpliTaq reaction buffer (Applied Bio-systems, Foster City, California, USA) containing 2.5 units of Taq polymerase, 200 µM each dNTP, 100 ng of forward and reverse primers, and 1.5 µl template from RT-PCR. After an initial denaturation stage of 95 C for 5 min, samples were run through 35 cycles at 95 C for 1 min, 55 C for 30 sec and 72 C for 1 min, followed by a final extension stage at 72 C for 5 min. The PCR products were analyzed by electrophoresis in 1–1.4% agarose gels.
The prevalence of antibodies to hanta-virus PUUV among the 65 Microtus voles was 6% (Table 1
). Of the IFA-positive voles, one Microtus guentheri lydius was found from the Izmir area, and one Microtus roberti and two Microtus rossiaemeridionalis from the Trabzon area (Table 1). The 264 Apodemus spp. screened for SAAV antibodies and the one D. nitedula screened for PUUV and SAAV antibodies tested negative. All rodents were screened for CPXV antibodies, but only one CPXV-IFA-positive Apodemus sylvaticus (prevalence in this host species 1%) was detected from Trabzon (Table 1). Antibodies against arenaviruses were found in eight (2.4%) of the tested 330 rodents (Table 1) and from all study sites. PCR results from rodents testing positive for antibodies to hantavirus (n = 4) and arenavirus (n = 8) were negative. Both CPXV (n = 330) antibody-positive and -negative samples were tested by PCR with negative results.
The presence of hantavirus antibodies in rodents from Turkey was demonstrated for the first time. Attempts to further characterize the hantavirus from the three species of Microtus voles by PCR were not successful. In Eurasia, hantaviruses are carried by several, but not all, Microtus species (Nemirov et al., 2004). In central and eastern Europe, TULV is widespread in Microtus arvalis, but this virus has not been unequivocally associated with human disease (Vapalahti et al., 2003). The antibody-positive rodents were trapped from regions where human cases have been reported (Kavukcu et al., 1997), and this association may warrant additional study. In contrast to reported results from wild mice from eastern and central Europe (Vapalahti et al., 2003), none of the Apodemus in this study were hantavi-rus seropositive. The reason for this is not known.
Only one CPXV-IFA-positive rodent (A. sylvaticus) was detected in this study, even though all samples were tested by IFA and PCR. There is no previous information on the occurrence of poxviruses in Turkish rodents. Elsewhere, a high prevalence of CPXV has been detected in voles, especially during fall (Chantrey et al., 1999; Pelkonen et al., 2003). The timing of our rodent collections (spring) as well as the low number of voles (n = 65) tested may explain the low prevalence of antibodies and infection observed in this study.
An intriguing finding in our recent surveys of rodents in different parts of Europe (Kallio-Kokko et al., 2005; Laakkonen et al., 2006) has been the relatively high prevalence of LCMV positive rodents. In this study, arenavirus-seropositive animals were found from all study sites (Table 1). In Europe, LCMV prevalence rates (based on antibody detection) found in the primary reservoir species, M. musculus, have ranged from 3.6% to 11.7% (Ackermann et al., 1964; Lledó et al., 2003); outside of Europe prevalence rates ranging from 2.5% to 9% have been reported (Morita et al., 1991; Childs et al., 1992). Mus musculus were not captured in this study, but the prevalence of antibodies to LCMV in Apodemus spp. ranged from 1% to 10%, and it was even higher in voles (1–13%). There are no previous studies on arenaviruses of rodents from Turkey. Our finding of arenavirus antibodies in voles raises the question whether members of the arvicoline subfamily carry a unique arenaviruses or whether these infections represent a spillover of arena-viruses normally associated with mice. Unfortunately, our attempts to characterize the viruses were unsuccessful; the identity of arenaviruses infections in wild rodents in Turkey awaits isolation of these viruses from the various host species.
Our work was supported by the European Union grants EU-QLR2-CT-2002-01358 and EDEN. This publication has been partially funded under the EU 6th Framework Program (GOCE-CT-2003-010284 EDEN) and is officially catalogued by the EDEN Steering Committee as EDEN0005. Its content does not represent the official position of the European Commission and is entirely the responsibility of the authors. We thank Tytti Manni and Auli Saarinen for superb technical assistance.
7 These authors contributed equally to this paper 
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Received for publication 18 May 2005.
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