Inkoo And Sindbis Viruses In Semi-Domesticated Reindeer And Mosquitoes In Norway

Background: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway because there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of SINV and INKV in mosquitoes and seroprevalence of INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). Methods: In total, 213 pools containing about 25 mosquitoes each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013-2015. The mosquito pools were analysed for INKV and SINV RNA, with reverse transcriptase (RT)-real time quantitative PCR (RT-qPCR), and pyrosequencing. Reindeer sera were analysed for INKV-specic IgG by Indirect Immunouorescence Assay (IIFA) and Plaque Reduction Neutralization Test (PRNT). Results: Aedes spp. were the most dominant species among the collected mosquitoes. Two of the mosquito pools were positive for INKV-RNA by RT-PCR and were conrmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. IgG seroprevalence in reindeer revealed 60% positive for INKV by IIFA. Of the 55 borderline reindeer sera, 24% were positive on cytopathic effect (CPE)-neutralization test. Among 80% of 60 reindeer sera analysed with PRNT for INKV had a titre ≥ 20, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snow Shoe Hare virus (SSHV). None of the analysed mosquito pools were positive for SINV. Conclusions:


Background
Mosquito-borne viruses emerge as a group that poses a serious threat to human health worldwide [1]. There has been an upsurge in the number of mosquito-borne viruses in Europe mostly belonging to the families Togaviridae (Sindbis virus SINV, Chikungunya virus), Flaviviridae (West Nile virus, Dengue virus), and Peribunyaviridae (Inkoo virus INKV, Batai virus BATV, Tahyna virus TAHV) [2,3].
Surveillance of mosquito-borne viruses and their prevalence in the human and animal populations is highly required since INKV, SINV, Chikungunya virus and West Nile virus infections often are not reported and remain undiagnosed [2]. Among other mosquito-borne viruses, INKV and SINV are known to be circulating in Norway, Sweden [4,1,5], Finland, and Russia, [6, 7,8,9]. In some parts of Russia INKV and SINV are associated with human morbidities. In humans, INKV causes mild fever to fatal encephalitis while SINV causes arthritis and rashes [10,11].
SINV is a member of the Western equine encephalomyelitis virus complex. It is an enveloped, positive sense, single-stranded (ss) 11.7-kb (kilo-base pair) RNA virus belonging to the genus Alphavirus in the Togaviridae family. Wild birds are considered as virus reservoir [18]. Antibodies against SINV have been detected in migratory and residential birds [19,20]. Ornithophilic mosquitoes (mosquitoes that feed on birds) of the genera Culex (Cx.) and Culiseta as well as Ochlerotatus spp. and Aedes (Ae.) spp. are considered to be the major SINV vectors [2]. Experimental infection studies implicate Cx. torrentium as a competent vector for SINV in northern Sweden [21,22,23]. The vector species of SINV in Sweden prefer lowland forested wetlands and humid forests with deciduous and coniferous trees as habitats [19]. SINV causes human arthritic diseases [24,25], known by various names in Scandinavia; "Baerplukkersyken" in Norway [26], Ockelbo disease in Sweden, [27], Karelian fever in Russia [28] and Pogosta disease in Both INKV and SINV have been isolated from diverse species of mosquitoes including Ae. cinereus, Cx. torrentium, Cx. pipiens, Culiseta morsitans, Ae. communis and Ae. punctor [8,29]. Moreover, evidence pointing to vertical transmission of these viruses in vectors has been demonstrated through detection of viral RNA in mosquito larvae [1]. In addition, SINV has been suggested to overwinter in Cx. pipiens mosquitoes [30]. Antibodies against INKV and SINV have been detected in human populations in Sweden and Finland [31,32,33]. In Russia, chronic neurological disease was reported in patients with antibodies to INKV [34]. Acute human INKV infections in association with encephalitis have been reported in Finland [35].
In Norway, INKV and SINV have not been studied extensively despite ndings of viruses in humans and mosquitoes in 1978 and 1992 [7,36,37]. There is a lack of knowledge covering the distribution and prevalence of these viruses in mosquito vectors, humans, potential reservoirs, and the regions affected. Other California Encephalitis (CE) group viruses have previously been isolated in Norway, but the methods used to identify them were not speci c enough to con rm if it was INKV or related viruses [16]. There are several cases of encephalitis with unknown etiology reported to Norwegian Surveillance System for Communicable Diseases (MSIS) [26]. These may be associated with INKV or other vector-borne pathogens. According to the Norwegian Institute of Public Health (2013), SINV cases are not reported to the MSIS.
A recent study indicated the need of monitoring INKV in vertebrate hosts such as moose and reindeer to identify the possible risk of transmission to humans [29]. The present study aimed at providing new knowledge on the prevalence and distribution of INKV and SINV in mosquitoes, and to investigate their presence in semi-domesticated reindeer in Norway. The combined analysis of mosquito and reindeer samples provides an indication of the distribution of these viruses in Norway, which could be addressed further and evaluated as potential causes of human encephalitis cases in Norway.

Methods
Study area and collection of samples  (Table 1, Fig. 1). To cover a variety of different species the sampling was performed at the reindeer summer pastures twice during the summer season. Mosquitoes were caught with a mosquito trap (Mosquito Magnet Independence; Woodstream ® Corporation, Pennsylvania, USA), using propane gas to produce carbon dioxide (CO 2 ) and a biting insect attractant (Mosquito Magnet Octenol, Woodstream ® Corporation, Pennsylvania, USA). The trap was set to run for two to 24 h depending on the abundance of mosquitoes and weather conditions. Mosquitoes were killed by freezing and stored at -20 °C from two days and up to two weeks after capture, until a -80 °C freezer was available. The mosquitoes from each collection site were separated into different cryotubes (Thermo Fisher Scienti c, Rochester, NY, USA). The mosquitoes were stored in a 50 ml Falcon tube (Corning Science Mexico, Reynosa, Mexico) with Drierite desiccant to dehydrate and preserve the samples for visual inspection and species identi cation [38]. All the cryotubes were stored at -80 °C until further analysis.  (Table 1). These animals were grazing on the same summer pastures as the mosquitoes were collected. Blood was obtained from live animals by venepuncture of external jugular vein using a venoject needle (Terumo, Leuven, Belgium) and blood collecting tubes (BD Vacutainer ® ; BD, Plymouth, UK). From slaughtered animals, blood was collected directly in the blood collecting tubes when bled. Blood tubes were centrifuged at 3500 rpm for 10 min to collect the serum. Sera were stored at -20 °C until analysis.

Extraction of total nucleic acid from mosquitoes
Each of the 213 pools of mosquito samples were transferred to 2 ml tubes containing six steel beads (MP Biomedical Life Science, CA, USA) and 350 ml of RLT® lysis buffer with β-mercaptoethanol in a ratio of Included on each plate was SINV RNA positive control, from the Ockelbo strain, cultured in the lab, in a 10fold serial endpoint dilution (10 -4 to 10 -7 ) along with two RNase free water samples as negative control.

Pyrosequencing
All the PCR positive samples for INKV RNA were further analysed by Pyrosequencing according to [41], for SQA analysis with the BioTage (Pyromark Q24) system (QIAGEN, Hilden, Germany). A 10-fold serial endpoint dilution (10 -1 to 10 -4 ) of INKV positive controls and RNase free water as negative control were included in each test.
The positive controls were used as a standard to compare the sequences obtained from pyrosequencing of PCR positive samples.

Calculation of prevalence
Two hundred and thirteen pools with approximately 25 mosquitoes in each pool were analysed. The sample size was designed for other research purposes by the CARD project. Estimated pooled prevalence (EPP) of INKV was calculated by the Epitools Epidemiological calculators [42]. The estimated prevalence was assumed to be close to zero with 95% con dence interval (CI) using Method 2 [43]. This method utilizes the frequentists approach assuming a xed pool size and perfect (100%) test sensitivity and speci city for the estimation of prevalence and con dence limits [41].

Cytopathic effect neutralization test (CPE-NT) from reindeer sera
Approximately 55 of the borderline reindeer sera samples were further veri ed by the CPE-neutralization test at the Department of Virology, University of Helsinki as previously described [33].

Plaque Reduction Neutralization Test (PRNT)
Reindeer sera (n = 66) with strongly positive, borderline, and negative result via IIFA were con rmed with a neutralization assay to determine whether the IgG antibodies were speci c to INKV, TAHV, or SSHV as previously described [32]. A 50% plaque reduction neutralization titer (PRNT 50 ) was calculated as the reciprocal of the highest serum dilution based on 50% or greater reduction in the plaque counts. Samples with titres ≥20 were de ned as INKV seropositive.
Statistical method for calculation of seroprevalence   Table 2). One of the positive pools had 70% while the other had 100% sequence similarity to the INKV positive controls (10 −1 to 10 −4 ) in each run (data not shown). The two positive pools were from Fosen and Røros from 2014. From this, the overall EPP was calculated to be 0.04% by the Epitools Ausvet calculator [42], with a site EPP of 0.5%.      Of the seropositives, 39% of the reindeer were female, 23% and male and 2% of unknown gender ( Table   3). Most of the female that were seropositives were adults (21%) while 17% of male calves were seropositive. The highest (60%) seropositives were female calves from Fosen 2014 followed by 55% of female adults from Tana 2013. The lowest seropositivity (4%) was among male adults and female calves from Lødingen 2013, and female adults from Valdres 2013.

Cytopathic effect neutralization test (CPE-NT) from reindeer sera
Of the 55 borderline reindeer sera samples from IIFA, 42 (76%) of the samples were con rmed to be negative and had a titre 20 while 13 (24%) of the remaining samples were positive on neutralization test (data not shown).

Discussion
The frequent movement of humans and animals across countries might contribute to spread viruses and vectors. Currently, there is no INKV surveillance in Norway. As part of the surveillance of viral infections that may cause encephalitis, we analysed reindeer sera as sentinel animals for the surveillance of Rjukan is situated south of the southernmost collection sites in our study (Valdres). Since there were low numbers of Cx. in the present study we cannot rule out presence of SINV in the studied areas. Also, the presence of migratory birds could affect incidence of SINV. However, SINV could not be detected from the collected mosquito samples by RT real-time PCR in the present study. Increasing the sample size of the mosquito pools may also have an in uence on the possibility to detect positives.
The ndings in this study are important for public awareness about geographical distribution of the virus. This knowledge may help to take necessary preventive measures against INKV infections in the future. In further studies it would be interesting to screen mosquitoes and the human population to determine the seroprevalence of INKV during the mosquito season, as well as whole genome sequencing of both isolated INKV to identify which strains are circulating in Norway.

Conclusions
There are knowledge gaps regarding the occurrence, distribution, and prevalence of the mosquito-borne viruses, INKV and SINV in Norway. The last study on these viruses was in 1992, reporting existence of these viruses in the country. Our study was important to establish a baseline and provide new knowledge on the prevalence and distribution of INKV and SINV in mosquitoes and reindeer populations. Moreover, it is important to monitor these viruses in case of an outbreak, and to avoid undiagnosed and unreported human cases. This study indicates that there is a low prevalence of INKV in mosquitoes in Norway. The Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests
No competing nancial interests exist.

Funding
This study was funded by the Norwegian Ministry of Health and Care Services, Barents region project B1710 "Emerging infections. Capacity building on vector-borne infections in the Barents region, Norway