Current and future environmental suitability for bats hosting potential zoonotic pathogens in rural Kenya.

Synanthropic bats live in close proximity to humans and domestic animals, creating opportunities for potential pathogen spillover. We explored environmental correlates of occurrence for a widely distributed synanthropic African bat, Mops pumilus-a species associated with potential zoonotic viruses-and estimated current and future environmental suitability in the Taita Hills region and surrounding plains in Taita-Taveta County in southeast Kenya. To project future environmental suitability, we used four Coupled Model Intercomparison Project Phase 6 general circulation models that capture temperature and precipitation changes for East Africa. The models were parameterized with empirical capture data of M. pumilus collected from 2016 to 2023, combined with satellite-based vegetation, topographic, and climatic data to identify responses to environmental factors. The strongest drivers for current environmental suitability for M. pumilus were short distance to rivers, higher precipitation during the driest months, sparse vegetation-often related to urban areas-and low yearly temperature variation. To predict current and future areas suitable for M. pumilus, we created ensemble niche models, which yielded excellent predictive accuracies. Current suitable environments were located southward from the central and southern Taita Hills and surrounding plains, overlapping with urban centers with the highest human population densities in the area. Future projections for 2050 indicated a moderate increase in suitability range in the southern portion of the region and surrounding plains in human-dominated areas; however, projections for 2090 showed a slight contraction of environmental suitability for M. pumilus, potentially due to the negative impact of increased temperatures. These results show how environmental changes are likely to impact the human exposure risk of bat-borne pathogens and could help public health officials develop strategies to prevent these risks in Taita-Taveta County, Kenya, and other parts of Africa.

Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

Complete coding sequence of an Aedes flavivirus strain isolated from Aedes albopictus collected in Northern Italy.

Complete genome data for the globally distributed Aedes flavivirus (AEFV) is scarce. We analyzed a new Italian AEFV strain isolated from Aedes albopictus. The results demonstrated genetic diversity among Italian AEFVs. The high similarity between AEFV genomes across geographically distant regions suggests long distance spreading via invasive host mosquito species.

SARS-CoV-2 infections among pregnant women, 2020, Finland-Cross-testing of neutralization assays.

We studied the development of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) pandemic in southern Finland in 2020 and evaluated the performance of two surrogate immunoassays for the detection of neutralizing antibodies (NAbs). The data set consisted of 12 000 retrospectively collected samples from pregnant women in their first trimester throughout 2020. All the samples were initially screened for immunoglobulin G (IgG) with SARS-CoV-2 spike antibody assay (EIM-S1, Euroimmun) followed by confirmation with nucleocapsid antibody assay (Architect SARS-CoV-2, Abbott). Samples that were reactive (positive or borderline) with both assays were subjected to testing with commercial surrogate immunoassays of NeutraLISA (EIM) and cPassTM (GenScript Biotech Corporation) by using pseudoneutralization assay (PNAbA) as a golden standard. No seropositive cases were detected between January and March. Between April and December, IgG (EIM-S1 and Abbott positive) and NAb (PNAbA positive) seroprevalences were between 0.4% and 1.4%. NeutraLISA showed 90% and cPass 55% concordant results with PNAbA among PNAbA negative samples and 49% and 92% among PNAbA positive samples giving NeutraLISA better specificity but lower sensitivity than cPass. To conclude, seroprevalence in pregnant women reflected that of the general population but the variability of the performance of serological protocols needs to be taken into account in inter-study comparison.

Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland.

BACKGROUND: Ticks carry microbes, some of which are pathogenic for humans and animals. To assess this One Health challenge, 342 ticks were collected from pet dogs and cats at 10 veterinary clinics in Finland as part of the European project "Protect Our Future Too". METHODS: The tick species were identified, and ticks were screened with quantitative PCR (qPCR) for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Ehrlichia canis, Anaplasma spp., Candidatus Neoehrlichia mikurensis, tick-borne encephalitis virus (TBEV), and Babesia spp. For comparison, a subset of tick DNA (20 qPCR-positive samples) was analysed with 16S next-generation sequencing (NGS). RESULTS: Most ticks were Ixodes ricinus (289, 84.5%), followed by Ixodes persulcatus (51, 14.9%). One hybrid tick (I. ricinus/I. persulcatus, 0.3%) and one Rhipicephalus sanguineus tick (0.3%) were identified. We found one or more of the analysed pathogens in 17% (59/342) of the ticks. The most prevalent pathogen was B. burgdorferi s.l. (36, 10.5%), followed by Anaplasma phagocytophilum (12, 3.5%), B. miyamotoi (5, 1.5%), Babesia venatorum (4, 1.2%), and TBEV (1, 0.3%). Candidatus Neoehrlichia mikurensis DNA was amplified from three (0.9%) ticks. Ehrlichia canis was not detected. In the 16S NGS, six samples produced enough reads for the analysis. In these six samples, we confirmed all the positive qPCR findings of Borrelia spp. and Ca. N. mikurensis. CONCLUSIONS: The high prevalence of pathogenic microorganisms in the ticks of this study emphasizes the importance of awareness of ticks and tick-borne diseases and prevention. Furthermore, the results show that veterinary surveillance can facilitate early detection of tick-borne pathogens and new tick species and draw attention to possible co-infections that should be considered both in symptomatic humans and animals after tick bites.

Vector Competence of Northern European Culex pipiens Biotype pipiens and Culex torrentium to West Nile Virus and Sindbis Virus.

The West Nile Virus (WNV) and Sindbis virus (SINV) are avian-hosted mosquito-borne zoonotic viruses that co-circulate in some geographical areas and share vector species such as Culex pipiens and Culex torrentium. These are widespread in Europe, including northern parts and Finland, where SINV is endemic, but WNV is currently not. As WNV is spreading northwards in Europe, we wanted to assess the experimental vector competence of Finnish Culex pipiens and Culex torrentium mosquitoes to WNV and SINV in different temperature profiles. Both mosquito species were found susceptible to both viruses and got infected via infectious blood meal at a mean temperature of 18 °C. WNV-positive saliva was detected at a mean temperature of 24 °C, whereas SINV-positive saliva was detected already at a mean temperature of 18 °C. Cx. torrentium was found to be a more efficient vector for WNV and SINV over Cx. pipiens. Overall, the results were in line with the previous studies performed with more southern vector populations. The current climate does not seem optimal for WNV circulation in Finland, but temporary summertime transmission could occur in the future if all other essential factors are in place. More field data would be needed for monitoring and understanding the northward spreading of WNV in Europe.

Stable Levels of Antibodies Against Unrelated Toxoid Vaccines After COVID-19: COVID-19 Infection Does Not Affect Toxoid Vaccine Antibody Levels.

Background: Lymphopenia is common in COVID-19. This has raised concerns that COVID-19 could affect the immune system akin to measles infection, which causes immune amnesia and a reduction in protective antibodies. Methods: We recruited COVID-19 patients (n = 59) in Helsinki, Finland, and collected plasma samples on 2 to 3 occasions during and after infection. We measured IgG antibodies to diphtheria toxin, tetanus toxoid, and pertussis toxin, along with total IgG, SARS-CoV-2 spike protein IgG, and neutralizing antibodies. We also surveyed the participants for up to 17 months for long-term impaired olfaction as a proxy for prolonged post-acute COVID-19 symptoms. Results: No significant differences were found in the unrelated vaccine responses while the serological response against COVID-19 was appropriate. During the acute phase of the disease, the SARSCoV-2 IgG levels were lower in outpatients when compared to inpatients. SARS-CoV-2 serology kinetics matched expectations. In the acute phase, anti-tetanus and anti-diphtheria IgG levels were lower in patients with prolonged impaired olfaction during follow up than in those without. Conclusions: We could not detect significant decline in overall humoral immunity during or after COVID-19 infection. In severe COVID-19, there appears to be a temporary decline in total IgG levels.

Predicting habitat suitability for Ixodes ricinus and Ixodes persulcatus ticks in Finland.

BACKGROUND: Ticks are responsible for transmitting several notable pathogens worldwide. Finland lies in a zone where two human-biting tick species co-occur: Ixodes ricinus and Ixodes persulcatus. Tick densities have increased in boreal regions worldwide during past decades, and tick-borne pathogens have been identified as one of the major threats to public health in the face of climate change. METHODS: We used species distribution modelling techniques to predict the distributions of I. ricinus and I. persulcatus, using aggregated historical data from 2014 to 2020 and new tick occurrence data from 2021. By aiming to fill the gaps in tick occurrence data, we created a new sampling strategy across Finland. We also screened for tick-borne encephalitis virus (TBEV) and Borrelia from the newly collected ticks. Climate, land use and vegetation data, and population densities of the tick hosts were used in various combinations on four data sets to estimate tick species' distributions across mainland Finland with a 1-km resolution. RESULTS: In the 2021 survey, 89 new locations were sampled of which 25 new presences and 63 absences were found for I. ricinus and one new presence and 88 absences for I. persulcatus. A total of 502 ticks were collected and analysed; no ticks were positive for TBEV, while 56 (47%) of the 120 pools, including adult, nymph, and larva pools, were positive for Borrelia (minimum infection rate 11.2%, respectively). Our prediction results demonstrate that two combined predictor data sets based on ensemble mean models yielded the highest predictive accuracy for both I. ricinus (AUC = 0.91, 0.94) and I. persulcatus (AUC = 0.93, 0.96). The suitable habitats for I. ricinus were determined by higher relative humidity, air temperature, precipitation sum, and middle-infrared reflectance levels and higher densities of white-tailed deer, European hare, and red fox. For I. persulcatus, locations with greater precipitation and air temperature and higher white-tailed deer, roe deer, and mountain hare densities were associated with higher occurrence probabilities. Suitable habitats for I. ricinus ranged from southern Finland up to Central Ostrobothnia and North Karelia, excluding areas in Ostrobothnia and Pirkanmaa. For I. persulcatus, suitable areas were located along the western coast from Ostrobothnia to southern Lapland, in North Karelia, North Savo, Kainuu, and areas in Pirkanmaa and Päijät-Häme. CONCLUSIONS: This is the first study conducted in Finland that estimates potential tick species distributions using environmental and host data. Our results can be utilized in vector control strategies, as supporting material in recommendations issued by public health authorities, and as predictor data for modelling the risk for tick-borne diseases.

Sindbis virus outbreak and evidence for geographical expansion in Finland, 2021.

Sindbis virus (SINV) caused a large outbreak in Finland in 2021 with 566 laboratory-confirmed human cases and a notable geographical expansion. Compared with the last large outbreak in 2002, incidence was higher in several hospital districts but lower in traditionally endemic locations in eastern parts of the country. A high incidence is also expected in 2022. Awareness of SINV should be raised in Finland to increase recognition of the disease and prevent transmission through the promotion of control measures.

Characterisation of the RNA Virome of Nine Ochlerotatus Species in Finland.

RNA viromes of nine commonly encountered Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16-60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date.

Serological Evidence of Exposure to Onyong-Nyong and Chikungunya Viruses in Febrile Patients of Rural Taita-Taveta County and Urban Kibera Informal Settlement in Nairobi, Kenya.

Several alphaviruses, such as chikungunya (CHIKV) and Onyong-nyong (ONNV), are endemic in Kenya and often cause outbreaks in different parts of the country. We assessed the seroprevalence of alphaviruses in patients with acute febrile illness in two geographically distant areas in Kenya with no previous record of alphavirus outbreaks. Blood samples were collected from febrile patients in health facilities located in the rural Taita-Taveta County in 2016 and urban Kibera informal settlement in Nairobi in 2017 and tested for CHIKV IgG and IgM antibodies using an in-house immunofluorescence assay (IFA) and a commercial ELISA test, respectively. A subset of CHIKV IgG or IgM antibody-positive samples were further analyzed using plaque reduction neutralization tests (PRNT) for CHIKV, ONNV, and Sindbis virus. Out of 537 patients, 4 (0.7%) and 28 (5.2%) had alphavirus IgM and IgG antibodies, respectively, confirmed on PRNT. We show evidence of previous and current exposure to alphaviruses based on serological testing in areas with no recorded history of outbreaks.

Seroevidence of Zoonotic Viruses in Rodents and Humans in Kibera Informal Settlement, Nairobi, Kenya.

Rodents are known reservoir hosts for a number of pathogens that can spillover into humans and cause disease. These threats are likely to be elevated in informal urban settlements (i.e., slums), where rodent and human densities are often high, rodents live in close proximity to humans, and human knowledge of disease risks and access to health care is often limited. While recent research attention has focused on zoonotic risks posed by urban rodents in major cities around the world, informal urban settlements have received far less attention. Here we report on a study in which samples were collected from 195 commensal rodents and 124 febrile human patients in the Kibera informal settlement in Nairobi, Kenya (one of the largest informal urban settlements in the world). Using immunofluorescence assays, samples were screened for antibodies against common rodent-borne zoonotic virus groups, namely orthopoxviruses, arenaviruses, and hantaviruses. We detected antibodies against orthopoxviruses in rodents (4.1% positive) and antibodies in humans against orthopoxviruses, arenaviruses, and hantaviruses (4.8%, 3.2%, and 8.1% positive, respectively). No rodents had antibodies against arenaviruses or hantaviruses. These results provide strong evidence for the circulation of zoonotic viruses in rodents and humans in Kibera urban settlement, but discordance between viruses detected in host groups indicates that other species or taxa may also serve as reservoirs for these zoonotic viruses or that humans testing positive could have been exposed outside of the Kibera settlement. More broadly, this study highlights the threat posed by zoonotic viruses in informal urban settlements and the need to mitigate human exposure risks.

Predicting Spatial Patterns of Sindbis Virus (SINV) Infection Risk in Finland Using Vector, Host and Environmental Data.

Pogosta disease is a mosquito-borne infection, caused by Sindbis virus (SINV), which causes epidemics of febrile rash and arthritis in Northern Europe and South Africa. Resident grouse and migratory birds play a significant role as amplifying hosts and various mosquito species, including Aedes cinereus, Culex pipiens, Cx. torrentium and Culiseta morsitans are documented vectors. As specific treatments are not available for SINV infections, and joint symptoms may persist, the public health burden is considerable in endemic areas. To predict the environmental suitability for SINV infections in Finland, we applied a suite of geospatial and statistical modeling techniques to disease occurrence data. Using an ensemble approach, we first produced environmental suitability maps for potential SINV vectors in Finland. These suitability maps were then combined with grouse densities and environmental data to identify the influential determinants for SINV infections and to predict the risk of Pogosta disease in Finnish municipalities. Our predictions suggest that both the environmental suitability for vectors and the high risk of Pogosta disease are focused in geographically restricted areas. This provides evidence that the presence of both SINV vector species and grouse densities can predict the occurrence of the disease. The results support material for public-health officials when determining area-specific recommendations and deliver information to health care personnel to raise awareness of the disease among physicians.

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays.

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection.

Sindbis Virus Strains of Divergent Origin Isolated from Humans and Mosquitoes During a Recent Outbreak in Finland.

Sindbis virus (SINV) is a mosquito-borne avian hosted virus that is widely distributed in Europe, Africa, Asia, and Oceania. Disease in humans is documented mainly from Northern Europe and South Africa and associated with genotype I. In 2018 under extremely warm climatic conditions, a small outbreak of 71 diagnosed SINV infections was recorded in Finland. We screened 52 mosquito pools (570 mosquitoes) and 223 human sera for SINV with real-time RT-PCR and the positive samples with virus isolation. One SINV strain was isolated from a pool (n = 13) of genus Ochlerotatus mosquitoes and three strains from patient serum samples. Complete genome analysis suggested all the isolates to be divergent from one another and related to previous Finnish, Swedish, and German strains. The study provides evidence of SINV strain transfer within Europe across regions with different epidemiological characteristics. Whether these are influenced by different mosquito genera involved in the transmission remains to be studied.

A novel negevirus isolated from Aedes vexans mosquitoes in Finland.

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

Detection of dengue virus type 2 of Indian origin in acute febrile patients in rural Kenya.

Dengue virus (DENV) has caused recent outbreaks in coastal cities of Kenya, but the epidemiological situation in other areas of Kenya is largely unknown. We investigated the role of DENV infection as a cause of acute febrile disease in non-epidemic settings in rural and urban study areas in Kenya. Altogether, 560 patients were sampled in 2016-2017 in rural Taita-Taveta County (n = 327) and urban slums of Kibera, Nairobi (n = 233). The samples were studied for DENV IgM, IgG, NS1 antigen and flaviviral RNA. IgG seroprevalence was found to be higher in Taita-Taveta (14%) than in Nairobi (3%). Five Taita-Taveta patients were positive for flaviviral RNA, all identified as DENV-2, cosmopolitan genotype. Local transmission in Taita-Taveta was suspected in a patient without travel history. The sequence analysis suggested that DENV-2 strains circulating in coastal and southern Kenya likely arose from a single introduction from India. The molecular clock analyses dated the most recent ancestor to the Kenyan strains a year before the large 2013 outbreak in Mombasa. After this, the virus has been detected in Kilifi in 2014, from our patients in Taita-Taveta in 2016, and in an outbreak in Malindi in 2017. The results highlight that silent transmission occurs between epidemics and also affects rural areas. More information is needed to understand the local epidemiological characteristics and future risks of dengue in Kenya.

Immunoassay for serodiagnosis of Zika virus infection based on time-resolved Förster resonance energy transfer.

Zika virus (ZIKV) is a mosquito-borne pathogen causing a febrile illness with arthralgia, conjunctivitis and rash. The complications include Guillain-Barré syndrome, congenital brain and other abnormalities and miscarriage. The serodiagnosis of ZIKV infection is hampered by cross-reactivity with other members of the Flavivirus family, notably dengue (DENV). This report describes a novel serological platform for the diagnosis of ZIKV infection. The approach utilizes time-resolved Förster resonance energy transfer (TR-FRET) elicited by two chromophore-labeled proteins (a ZIKV antigen and a super-antigen) simultaneously binding to a given antibody molecule. The antigen used in the assay is ZIKV non-structural protein 1 (NS1) and the super-antigen is bacterial protein L. Three assay variants were developed: the first measuring all anti-ZIKV-NS1 antibodies (LFRET), the second measuring IgM and IgA (acute-LFRET) and the third measuring IgG (immunity-LFRET). The assays were evaluated with a panel of samples from clinical ZIKV cases in travelers (n = 25) and seronegative (n = 24) samples. DENV (n = 38), yellow fever (n = 16) and tick-borne-encephalitis (n = 20) seropositive samples were examined for assessment of flavivirus cross-reactivity. The diagnostic sensitivities of the respective LFRET assays were 92%, 100% and 83%, and the diagnostic specificities 88%, 95% and 100% for LFRET, acute-LFRET and immunity-LFRET. Furthermore, we evaluated the assays against a widely-used commercial ELISA. In conclusion, the new FRET-based serological approaches based on NS1 protein are applicable to diagnosing zika virus infections in travelers and differentiating them from other flavivirus infections.

Bombali Virus in Mops condylurus Bat, Kenya.

Bombali virus (genus Ebolavirus) was identified in organs and excreta of an Angolan free-tailed bat (Mops condylurus) in Kenya. Complete genome analysis revealed 98% nucleotide sequence similarity to the prototype virus from Sierra Leone. No Ebola virus-specific RNA or antibodies were detected from febrile humans in the area who reported contact with bats.

Validation of serological and molecular methods for diagnosis of zika virus infections.

The laboratory confirmation of Zika virus (ZIKV) infection, and the differential diagnosis from other flavivirus infections such as dengue virus (DENV), often requires the use of several diagnostic test types. Cross-reactions and secondary infections complicate the serological diagnosis and specific viral RNA detection assays are often needed for confirming the diagnosis. The aim of this study was to validate serological and molecular methods for diagnosing ZIKV infection. This included the evaluation of a ZIKV RT-qPCR assay for diagnostics that was previously set up for research use and to compare the ZIKV, DENV and TBEV EIA methods. External and in-house controls and pre-characterized sample panels were tested, and also automated and manual nucleic acid extraction methods were compared. A total of ten Finnish traveler patients were diagnosed with acute ZIKV infection during 2015-2017 including one suspected dual DENV and ZIKV infection. These samples along with panels of DENV and tick-borne encephalitis virus (TBEV) infections were used to test the cross-reactive properties of ZIKV, DENV and TBEV IgM assays. Additionally, the diagnosed acute ZIKV patient samples were tested using commercially available diagnostic DENV NS1 antigen assay and a ZIKV NS1 antigen assay intended for research use. The ZIKV RT-qPCR assay was demonstrated to be both specific and sensitive (one genome per reaction) and suitable for routine diagnostic use utilizing automated nucleic acid extraction. Of the tested IgM tests the NS1 antigen-based ZIKV IgM (Euroimmun) assay performed with least cross-reactivity with a specificity of 97.4%. The DENV IgM assay (Focus Diagnostics) had specificity of only 86.1%. The results are in line with previous studies and additionally highlight that also acute TBEV patients may give a false positive test result in DENV and ZIKV IgM assays.