Abstract
Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are mainly transmitted by arthropod vectors to vertebrate hosts including humans, resulting in fever and neurological signs. The aim of this study was to investigate the presence of antibodies to TBEV and WNV, and TBEV-RNA and WNV-RNA in Turkish children with fever and/or arthritis. For this purpose, 110 sera and buffy-coat samples were collected; sera were analyzed by indirect enzyme-linked immunosorbent assay for the presence of IgM and IgG antibodies to TBEV and WNV, and buffy-coat-derived white blood cells were analyzed by quantitative real-time RT-PCR for TBEV-RNA and WNV-RNA. IgM antibodies to TBEV were detected in five children between the ages of 3 and 7 years; no IgG antibodies to TBEV were detected. IgG antibodies to WNV were detected in two children and IgM antibodies to WNV were detected in six children, between the ages of 3 and 7 years. One of the children had IgM antibodies to WNV and to TBEV. Children who had antibodies to TBEV and WNV had fever and/or arthritis but no obvious neurological signs. Molecular diagnostic approaches revealed that neither TBEV-RNA nor WNV-RNA was present in any of the buffy-coat samples, not even in children with IgM-specific antibodies. Our serological results indicate that children in Turkey are exposed to TBEV and WNV.
Introduction
Tick-borne encephalitis (TBE) and West Nile fever (WNF) are zoonotic vector-borne diseases affecting people and mammals worldwide. The etiological agent of TBE is TBE virus (TBEV), whereas the etiological agent of WNF is West Nile virus (WNV). Both viruses are enveloped single-stranded positive-sense RNA viruses belonging to the genus Flavivirus within the family Flaviridae (Klaus et al. 2010, Süss 2011, Chancey et al. 2015).
TBEV is transmitted by Ixodes spp. The main hosts are small rodents, with humans being accidental hosts (Süss 2011). Humans are infected through the bite of infected ticks, crushing of ticks by hand, and direct contact with tissues or body fluids (including milk of ruminants) of viremic animals and humans (Klaus et al. 2010, Bogovič and Strle 2015). WNV is mainly transmitted by Culex mosquitos and sometimes by blood transfusion, organ transplantation, laboratory practices, and in utero. Birds are the amplifying hosts, whereas horses, humans, and other mammals are dead end hosts (Chancey et al. 2015).
TBEV can cause disease in humans and a variety of animals such as fever, fatigue, and neurological signs due to meningitis, meningoencephalitis, or encephalomyelitis (Süss 2011, Bogovič and Strle 2015). The major symptoms of WNF infections are fever, influenza-like symptoms, and neurological disease in 1% of patients including acute flaccid paralysis, meningoencephalitis, encephalitis, and meningitis (Chowers 2001).
It is rather difficult to diagnose TBEV and WNV infections in humans and animals based on clinical signs, since the symptoms are often unspecific. Therefore, laboratory confirmations are critical. They are based on results of serology, virus isolation, and molecular tests (Saksida et al. 2005, Klaus et al. 2010). In this study, we aimed to investigate the presence of serological and molecular markers for TBEV and WNV in Turkish children with fever—a hallmark of TBEV and WNF infection—and/or arthritis.
Materials and Methods
Study population and sampling
In this study, a total of 110 children with arthritis and/or fever between 2 and 18 years old were examined during 2013–2016. Sera from these patients were analyzed for the presence of IgG and IgM antibodies to TBEV and WNV and for the presence of TBEV-RNA and WNV-RNA. For this purpose, blood was drained into plain tubes for enzyme-linked immunosorbent assay (ELISA) testing and tubes containing EDTA for quantitative real-time RT-PCR testing. All samples were transported to the laboratory in cold storage.
Enzyme-linked immunosorbent assay
IgG and IgM antibodies to TBEV and WNV were investigated by using commercial ELISA kits (Euroimmun-EI 2661-9601G; Euroimmun-2662-9601G; Euroimmun-EI 2661-9601M; Euroimmun-2662-9601M) as described by the manufacturer (Euroimmun, Germany). The sensitivity and specificity of the IgG and IgM ELISA for TBEV are 100% (data obtained from Euroimmun). The sensitivity and specificity of the IgG ELISA for WNV are 99.5% and 96.9%, respectively, and those for the IgM ELISA are 94% and 99.8%, respectively (data obtained from Euroimmun). Statistical analysis was applied to ELISA results by using the Fisher exact test.
In case of seropositivity to TBEV or WNV, these samples were analyzed for the presence of IgG antibodies to Crimean Congo hemorrhagic fever virus (CCHFV) by using a commercial ELISA kit (Vector Best, D-5052); the ELISA protocol followed the manufacturer's recommendations.
RNA extraction and cDNA synthesis
For the extraction of viral RNA from buffy-coat samples, QIAamp Viral RNA mini kit (Qiagen, Germany) was used. Extraction procedures were performed as described by the manufacturer. The amount of RNA in the extracted material was measured using a NanoDrop spectrophotometer (NanoDrop 1000c; Thermo Scientific). Reverse transcription was performed as described previously (Yilmaz et al. 2017). In brief, RT was performed in two steps. For the first step, 9 μL of RNA template (∼400 ng) was mixed with 1 μL random hexamers (Promega). For the second step, a total volume of 20 μL reaction mixture was prepared that consists of 10 μL RNA/primer mixture from the first step, 4 μL 5 × RT buffer, 2.4 μL 25 mM MgCl2, 1 μL 10 mM dNTPs, 1.6 μL nuclease-free water, and 1 μL reverse transcriptase (Improm II; Promega). After completion of the RT reaction (Yilmaz et al. 2017), 30 μL of nuclease-free water was added to each cDNA sample.
Quantitative real-time RT-PCR for detection of TBEV-RNA and WNV-RNA
Primers and probes used in this study were derived from previous published studies (Schwaiger and Cassinotti 2003, Linke et al. 2007). The methods and amplification conditions for the real-time PCR to detect TBEV-RNA and WNV-RNA in buffy-coat samples were the same as described previously (Schwaiger and Cassinotti 2003, Linke et al. 2007). Quantitative real-time PCRs were performed for the test samples in a 25 μL PCR by using positive and negative controls (Dr. Robert S. Lanciotti, Centers for Disease Control and Prevention, Dr. Christine Klaus, Friedrich-Loeffler-Institute, Germany) and amplification was performed in a Stratagene MX3005P real-time PCR instrument. For TBEV, an optimized PCR mixture consisted of 1 μL (50 nM) of forward and 1 μL (300 nM) of reverse primer, 1 μL (200 nM) probe, 12.5 μL Maxima Hot Start PCR Master Mix (K1052; Termo Scientific), 0.5 μL MgCl2, 4 μL of cDNA, and 5 μL nuclease-free water. The reaction conditions and cycles were the same as described by Schwaiger and Cassinotti (2003). For WNV, an optimized PCR mixture consisted of 2 μL (7.5 μM) of each forward and reverse primers, 1 μL (2.5 μM) probe, 12.5 μL Maxima Hot Start PCR Master Mix (K1052; Termo Scientific), 0.5 μL MgCl2, 5 μL of cDNA, and 2 μL nuclease-free water. The reaction conditions and cycles were the same as described by Linke et al. (2007).
In addition, seropositive samples for TBEV and WNV were also analyzed for the presence of CCHFV-RNA as described previously (Atkinson et al. 2012). A synthesized positive control was included in the 25 μL PCR mixture and PCR conditions and cycles were the same as indicated in a published study by Atkinson et al. (2012).
Results
Clinical status of patients
Physical examination revealed arthritis in 95 and fever and arthritis in 15 children. The 110 children with arthritis suffered from juvenile or rheumatoid arthritis, either as an acute or chronic form. The temperature measured in 15 children was between 38°C and 39.5°C.
Enzyme-linked immunosorbent assay
IgM antibodies to TBEV were detected in 5 out of 110 children between the age of 3 and 7 years; however, no IgG antibodies to TBEV were detected. The optical density (OD) values of the positive samples were 2.859, 1.014, 0.942, 0.726, and 0.651; the positive control antisera had an OD of 1.426. IgG antibodies to WNV were detected in 2 out of 110 children between the age of 3 and 7 years. The OD values of the positive samples were 1.242 and 0.997 with the positive control sera having an OD of 0.902. IgM antibodies to WNV were also detected in six children. The OD values of the positive samples were 3.506, 1.841, 1.350, 1.216, 1.182, and 1.175, and the positive control sera had an OD of 1.185. One child was IgM seropositive to both WNV and TBE. Children found to be seropositive for TBEV (n = 5) and WNV (n = 8) had arthritis as did the TBEV/WNV seronegative patients (n = 97). There was no association between seropositivity for TBEV and WNV and having arthritis (p > 0.05). Similarly, there was no association with having fever and TBEV/WNV seropositivity (p > 0.05).
Both TBEV and WNV seropositive children were living in Istanbul. Family of children who had antibodies to WNV were originally from the Black Sea, Inner Anatolia, and South East Anatolia. Interestingly, families of children seropositive for TBEV were originated from only the Central Black Sea region.
IgG antibodies to CCHFV were not detected in any of the children seropositive for TBEV and WNV. Children who had IgM or IgG antibodies to TBEV and WNV had fever and/or arthritis but no neurological signs. The children found seropositive to TBEV in our study had no history of tick bites according to their parents' observations. However, they may have been exposed to ticks but their parents may not have observed this.
Quantitative real-time RT-PCR
Buffy-coat-derived immune cells from all patients were tested for the presence of TBEV-RNA and WNV-RNA. Markers of TBEV or WNV infection were not detected in any of the buffy-coat samples. Target-specific amplication was only seen in our respective positive controls, but not in the negative controls. The CT value for the TBEV positive control was 29, for WNV 25. In addition, CCHVF-RNA was not detected in children who were seropositive for TBEV.
Discussion
TBE is an endemic disease in Europe, Siberia, Eastern Russia, Northern China, Japan, and the Republic of Korea (Süss 2011, Yoshii et al. 2017). Annually, 10,000–15,000 TBE cases are reported in Europe and Asia. In Europe, 27 countries reported endemic TBE with high incidences especially in Slovenia, Estonia, Lithuania, and Latvia. TBE is a notifiable disease in 16 European countries (Klaus et al. 2010, Bogovič and Strle 2015). According to a 2013 report, 15 TBE cases per 100,000 people were reported in Slovenia (Bogovič and Strle 2015). In Germany, the number of TBE risk areas has increased in the past 10 years, from 63 to 132 areas (Klaus et al. 2010).
Different seroprevalances were reported in different regions in Turkey concerning IgG and IgM antibodies to TBEV, and overall seroprevalance was found to be between 1.4% and 20.5% (Inci et al. 2016). In this study, 110 sera from children with arthritis and/or fever were investigated for the presence of TBEV. IgM antibodies to TBEV were detected in five (4.5%) children between the age of 3 and 7 years. No IgG antibodies to TBEV were detected. Frequency of IgM positivity found in this study is similar to that found previously in Turkish patients (Ergunay et al. 2012, Inci et al. 2016). In addition, analysis of the buffy-coat samples for the presence of TBEV-RNA was negative for all samples. Similar results were obtained in afformentioned studies within Turkey (Ergunay et al. 2012, Inci et al. 2016).
WNF cases have been reported in African countries, Europe, and America (Chancey et al. 2015). In the Mediterranean region, since 1960s, a number of outbreaks have been reported in Italy, Spain, and Greece (Di Sabatino et al. 2014). In Turkey, clinical cases were seen after 2009–2010 (Ocal et al. 2013), and antibodies to WNV varied between 0.9% and 20.4% (Ozkul et al. 2006, Ergunay et al. 2010, Ocal et al. 2013). In this study, IgG antibodies to WNV were detected in two (1.8%) and IgM antibodies to WNV in six (5.4%) children with fever and/or arthritis between the ages of 3 and 7 years. Both TBEV and WNV seropositive children were living in Istanbul. Families of children who had antibodies to WNV were originally from the Black Sea, Inner Anatolia, and Southeast Anatolia. Interestingly, families of children seropositive for TBEV were originated from only the Central Black Sea region. Children might have been infected either in Istanbul or in the city of family origin when they have visited grand parents. If the latest happened risk of being near or in the Black Sea region for TBEV, seropositivity cannot be excluded.
Children who had antibodies to TBEV and WNV had fever and/or arthritis but no neurological disorders. In this study, it is difficult to determine a correlation between WNV or TBEV seropositivity and patients with arthritis. However, future studies should focus on people with arthritis and their status regarding WNV and TBEV-specific markers. In addition, no TBEV-RNA or WNV-RNA was found in any of the buffy-coat samples, similar to other studies in Turkey (Ozkul et al. 2006, Ergunay et al. 2010). This could be due to either very low level of virus RNA in the samples tested or absence of viremia, which is rather short for both TBEV and WNV infections in humans (Holzmann 2003, Busch et al. 2008, Lustig et al. 2018).
According to Holzmann (2003), TBEV viremia is found during the initial phase of disease in most cases between 2 and 5 days postinfection. IgM antibody titers start to increase at the end of the initial phase and decline around 5 to 6 weeks after infection; a longer persistence can be observed in individual cases (Roggendorf et al. 1981, Holzmann 2003). Therefore, in most cases, TBEV-specific IgM serum antibody titers can be found after the initial TBEV viremic phase and not simultaneously. However, it cannot fully be excluded that TBEV viremia and IgM serum antibody titers occur simultaneously. In a study group in Slovenia (Saksida et al. 2005), a minor group of patients (23.1%) had a positive IgM serum antibody status and viremia (TBEV-RNA could be detected).
The average duration of WNV viremia has been reported in blood donors to be 6.9 days (Busch et al. 2008). As described for TBEV, WNV IgM antibody titers, and WNV viremia could occur simultaneously in few cases. However, WNV IgM antibody titers can persist for up to 1 year and residents in areas of endemicity can have persistent WNV IgM antibody titers unrelated to a concurrent illness (Fox et al. 2006, Lustig et al. 2018). It is assumed that because of the mentioned correlations, we were not able to detect TBEV/WNV-specific RNAs in IgM-positive patients.
The accuracy of positive and negative samples determined by the ELISA tests performed in this study depends on the sensitivity and specificity of the individual tests. The sensitivity and specificity of the IgG and IgM ELISA for TBEV are 100%. The sensitivity and specificity of IgG ELISA for WNV are 99.5% and 96.9%, respectively, whereas they were 94% and 99.8%, respectively, for the IgM ELISA. We assume that our ELISA results regarding TBEV reactivity were fairly accurate. In contrast, it is possible that very few sera tested for WNV could be false positive or false negative. The difficulty of serological diagnosis for flaviviruses was recently reviewed by Lustig et al. (2018).
In conclusion, results of these studies indicate that Turkish children are exposed to TBEV and WNV infection. No viral RNA of TBEV and WNV was detected. Children who had antibodies to TBEV and WNV had fever and/or arthritis but no obvious neurological signs. This is the first study reporting the prevalence of the TBEV and WNV infections in children with arthritis in Turkey.
Footnotes
Acknowledgments
This study was funded by the University of Istanbul (project no: 13163). The authors thank to Dr. Lancelotti for supplying positive WNV as PCR-positive control. “JAR: Center of Excellence for Emerging and Zoonotic Animal Diseases.”
Ethical Approval
Ethical approval was obtained from the Ethics Committee of the University of Istanbul.
Author Disclosure Statement
No competing financial interests exist.