A Review of Methods for Detecting Tick-Borne Encephalitis Virus Infection in Tick,Animal,and Human Specimens

Abstract

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted arbovirus causing human disease in Europe and Asia. Over the past decades, the incidence of TBEV infection has significantly increased, with over 13,000 annual hospital referrals in endemic countries and cases emerging in previously unaffected regions. Specific detection of TBEV is required to diagnose suspected human cases or during surveillance of tick vectors and/or susceptible animal species. Widely used techniques for diagnosis comprise serological methods to detect viral antigens or antibodies and nucleic acid tests to detect viral RNA in target specimens. Moreover, virus isolation using susceptible cell lines or vertebrates, electron microscopy, or immunohistochemistry can also be employed on specific occasions. The purpose of this review is to compile and outline various approaches and techniques for detecting TBEV infection in ticks, wild animals, and humans. Specific sections for specimen collection and storage, nucleic acid testing, and serological assays cover various aspects of dynamics, performance characteristics, and utility in the diagnostic workup of suspected cases. Impact of immunoglobulin M testing and quantification, immunoglobulin G avidity, and real-time and quantitative polymerase chain reaction methods were overviewed with assay comparisons. Recent advances in serological assays to mitigate the impact of cross-reactions were further discussed along with the detailed interpretation of laboratory test results in human infections.

Introduction

Tick-borne encephalitis (TBE), a life-threatening neurological disease caused by TBE virus (TBEV), is a growing public health concern in Europe and Asia (Süss 2011, European Centre for Disease Prevention and Control 2012, Lindquist 2014). TBEV is a member of the family Flaviviridae, genus Flavivirus, and has three subtypes; European (TBEV-Eu; formerly known as Central European encephalitis virus), Siberian (TBEV-Sib; formerly known as West Siberian virus), and Far Eastern (TBEV-FE; formerly known as Russian Spring Summer encephalitis virus) (Charrel 2004, Lindquist 2014). TBEV is maintained in ticks and their wild vertebrate hosts in forested natural foci. Ticks act as both the vector and reservoir for TBEV. Ixodes ricinus ticks are the main vector for TBEV-Eu, and Ixodes persulcatus is the dominant vector for TBEV-Sib and TBEV-FE. The main hosts are small mammals; large animals are feeding hosts for ticks, but do not play a role in the maintenance of the virus (Süss 2011, Estrada-Peña and de la Fuente 2014).

The epidemiology of TBE is closely associated with the ecology and biology of ticks and periods of their feeding activity. Human infection usually occurs as a result of tick bites, but the infection can also be transmitted via consumption of unpasteurized milk from infected animals (Charrel 2004, Süss 2011). The incubation period for TBE in humans can be from 2 to 28 days (usually between 7 and 14 days) and is asymptomatic. In most cases of TBE, a biphasic clinical course is observed. The first viremic stage is usually between 2 and 10 days and presents with influenza-like symptoms such as high fever, headache, or muscle pain. This is often followed by a symptom-free interval of about one week (range: 1–33 days) before the secondary neurological phase, which occurs in 20–30% of patients. The infection can manifest with different clinical forms (febrile, meningeal, meningoencephalitic, polyencephalitic, poliomyelitic, polioradiculoneuritic, etc.). TBE caused by viruses of different subtypes may vary not only in the frequency of different clinical forms, but also in the severity of each form (Růžek et al. 2010, Bogovic and Strle 2015).

This manuscript presents an overview of methods employed in the detection of TBEV infections in ticks, wild animals, and humans. Providing a complete list of all available techniques was not intended, but rather a more practical description of the frequently used approaches and strategies. Performance and comparison of methods as well as recommendations for sample collection techniques were discussed in detail wherever appropriate.

Besides applications in basic research, specific detection of TBEV is required in the diagnosis of suspected human cases or during surveillance of tick vectors or susceptible animal species. The arsenal of techniques available basically comprises serological methods to detect viral antigens or antibodies and nucleic acid tests to detect viral RNA in target specimens. Moreover, virus isolation using susceptible cell lines or vertebrates or electron microscopy of infected tissues can also be performed, despite being rarely employed for these purposes (Table 1).

Table 1.

Overview of Laboratory Techniques for Tick-Borne Encephalitis Virus Diagnosis

	Samples ^a					Performance
	Blood	Cerebrospinal fluid	Tissue	Milk	Tick	Advantages	Disadvantages
Virus isolation	+	(+)	+	+	+	Provides infectious virus for detailed characterization and further research.	Low sensitivity. Technically demanding. Appropriate biosafety conditions required.
Electron microscopy	−	−	+	−	+	Can be employed for direct visualization of the virus in infected tissues or cell culture.	Requires high number of virions. Technically demanding. Little or no impact on diagnosis.
Nucleic acid tests	+	(+)	+	+	+	High sensitivity and specificity. Quantification possible. Subtype detection possible. Partial sequence characterization possible.	Technically demanding. Relatively expensive. Extraction–purification step required. Limited diagnostic impact in late disease.
Serology
HI, CF	+	+	−	−	−	Historically used for diagnostics and characterization of antigenic relationships among viruses.	Increased antibody titers in acute and convalescent sera required for diagnosis. Group-specific antigens detected. Cross reactions among flaviviruses.
ELISA, IFA, IB	+	+	+^b	+	+^b	Rapid, less expensive, easy to perform. Single specimen can be employed for diagnosis. Quantification possible Antibody subtype determination possible (IgM, IgG, IgA). Antigen detection possible.	Cross-reactions among flaviviruses. Variations of sensitivity/specificity in commercially available assays.
VNT	+	+	−	+	−	Gold standard for specificity.	Technically demanding. Appropriate biosafety conditions required.
IHC	−	−	+	−	+	Can be employed for viral antigen detection in infected tissues.	Limited use in diagnostics.

+ or (+): reported or possible, −: not reported or improbable.

Antigen assays.

CF, complement fixation; ELISA, enzyme-linked immunosorbent assay; HI, hemaggltination inhibition; IB, immunoblot; IFA, immunofluorescence assay; Ig, immunoglobulin; IHC, immunohistochemistry; VNT, virus neutralization test.

Samples for TBEV Testing

TBEV nucleic acids can be detected in several clinical and environmental samples including serum, plasma, urine, tissue, and/or cerebrospinal fluid (CSF) samples from individuals with suspected TBEV-related diseases; ticks collected from the field for surveillance; and blood, brain, spleen, or other tissue samples from rodents/other animals suspected as reservoirs or in experimental infections. Moreover, inoculated cells or culture supernatants may be tested for TBEV nucleic acids during screening for virus replication, employing identical detection methods. Prior to the testing protocol, nucleic acid purification can be achieved via several standardized and widely used in-house or commercial assays (Donoso-Mantke et al. 2007). Commercially available extraction methods have been reported to perform better for correct virus characterization (Donoso-Mantke et al. 2007). Depending on the starting material, homogenization may be required for optimal nucleic acid extraction, especially when processing individual or pooled ticks and tissue specimens, for which several protocols have already been developed (Schwaiger and Cassinotti 2003, Achazi et al. 2011, Andreassen et al. 2012). The target nucleic acid type (RNA or DNA) and amount should be taken into consideration to obtain optimal outcomes in the nucleic acid purification step. Methods excluding the nucleic acid purification step via chelating agents to facilitate field surveillance have also been developed (Rudenko et al. 2004). For nucleic acid detection as well as virus isolation, the samples should be transported on dry ice, stored at −80°C, avoiding freeze–thaw cycles that may result in nucleic acid degradation and false negativity.

Serum, plasma, or whole blood specimens are sufficient for TBEV antigen or, more frequently, antibody detection in human and vertebrate samples via serological assays. Milk samples from farm animals can further be screened to assess potential transmission risks or virus exposure. The samples for antigen or antibody testing should be stored either refrigerated or frozen and with a possibility of long-term storage at −80°C. For serological assays, limiting freeze–thaw cycles is also recommended to avoid reduced antibody titers and false negativity.

For field sampling of ticks, standard methods called “dragging” and “flagging” are frequently employed. Dragging is based on moving a blanket fixed on a bar over vegetation. With a blanket of defined size, it is possible to calculate the surface of the screened area. All developmental stages of ticks can be collected using this method, but larvae and nymphs predominate (Donoso-Mantke et al. 2011). Flagging is done using a flag consisting of a stick approximately 1.5 meters long with a cloth fixed to the stick. Ticks are collected by moving the flag over the vegetation. The total number of ticks collected by flagging is usually higher compared with dragging, usually including more adults (Donoso-Mantke et al. 2011). Collected ticks can be stored temporarily alive in tubes with constant humidity at 4°C and can then be used for virus isolation or RNA extraction for nucleic acid detection. Tick specimens can be stored intact at −80°C, without freeze–thaw cycles, for further research. Ticks removed from suspected individuals should be processed (or frozen at −80°C) immediately, if virus screening is to be performed. There are several tick removal devices on the market, but a plain set of fine-tipped tweezers will remove ticks quite effectively.

TBEV screening in ticks can be performed on individual specimens and/or in pools. In pooled samples, the infection prevalence can be estimated via two frequently employed approaches; the minimum infection rate and the frequentist methods (Cowling et al. 1999, Ebert et al. 2010). The former is based on the assumption that a positive signal in a pool originates from a single infected tick and average minimum infection prevalence is provided, whereas in the latter maximum-likelihood estimates of true prevalence can be calculated with confidence limits. The pool size and content (whether adults, larvae, and nymphs are grouped or mixed) is also likely to influence the prevalence rates (Andreassen et al. 2012).

Assays for TBEV Testing

Virus isolation

Although rarely attempted for diagnostics or surveillance because of technical obstacles and low sensitivity compared with other diagnostic approaches, virus isolation remains the gold standard method for confirming a presence of a viable virus in the investigated sample. It also enables complete virus characterization and facilitates further research. TBEV isolation from human, vertebrate and tick vector specimens can be accomplished in properly collected, transferred, and stored specimens with a sufficient amount of viable virions. Postmortem brain or other tissue samples can also be employed for virus isolation and electron microscopy (Jellinger 1981). Isolation is generally attempted and successful in specimens with detectable viral RNA in high copy numbers, which frequently comprise field-collected ticks. Assays involving manipulation of live virus strains, such as isolation and virus neutralization assay (VNT) require an appropriate biocontainment facility—a biosafety level 3 facility in most Western countries (Holzmann 2003).

A wide spectrum of cell lines susceptible to TBEV, which include the porcine kidney stable cell line (Kožuch and Mayer 1975), Vero, HeLa, BHK-21, etc., are available for virus isolation. Following the inoculation of the tick or human specimen, cell cultures should be observed daily for the presence of a cytopathic effect (rounding of the infected cell, loss of integrity of the cell monolayer, fusion with adjacent cells to form syncytia, and cell death). In case of very low virus titers in the original specimen, the cytopathic effect can be weak or can develop only after repeated subcultures. Propagating virus can be detected in the inoculated cells by indirect immunofluorescence or in culture medium by PCR.

Suckling mice, which are highly sensitive to TBEV, can further be employed for virus isolation. Two- to six-day-old suckling mice are inoculated intracerebrally with approximately 0.01 mL of the sample, and eventually intraperitoneally with 0.03 mL. Inoculated mice should be monitored twice daily for up to 2 weeks for signs of illness (neurological signs, apathy or increased activity, mice out of the nest) and death. Brains of euthanized or dead mice with compatible symptoms are then removed and frozen at −80°C until further processing. Chicken embryos are also sensitive to TBEV but are rarely employed for virus isolation. The investigated material can be inoculated into the yolk sac or to the chorioallantoic membrane of 6- to 9-day-old chicken embryos (Slonim and Röslerová 1965, Slonim et al. 1966).

Nucleic acid tests

Nucleic acid tests (NATs) involve the detection of TBEV RNA and provide excellent sensitivity and specificity when optimal sample collection is warranted. As discussed previously, NATs require the pre-analytic step of nucleic acid purification. Purified viral RNA must further undergo a cDNA synthesis step via a reverse transcription reaction, unless a “one-step” protocol, frequently incorporated within a real-time PCR method, has been employed (Schwaiger and Cassinotti 2003, Achazi et al. 2011, Katargina et al. 2013). Standardized commercial kits have frequently been used for cDNA synthesis (Achazi et al. 2011, Paulsen et al. 2015). The reaction can be primed via randomized short oligonucleotides (provided commercially) or TBEV-specific primers of specific PCR protocols (Puchhammer-Stockl et al. 1995, Schrader and Süss 1999, Achazi et al. 2011, Andreassen et al. 2012). Randomized oligonucleotides provide the advantage of targeting various genomic regions and pathogen RNAs, while assay specificity can be enhanced with TBEV-specific primers.

Various genomic targets such as envelope (E) (Skarpaas et al. 2006, Růzek et al. 2007, Tkachev et al. 2008, Andreassen et al. 2012), nonstructural proteins NS1 (Achazi et al. 2011), NS3 (Katargina et al. 2013), and NS5 (Puchhammer-Stockl et al. 1995) (Table 2) and 5′ and 3′ untranslated regions (Schrader and Süss 1999, Schwaiger and Cassinotti 2003) are employed for PCR detection of TBEV. Several single-step and nested PCR protocols have been reported and tested for utility in tick screening and clinical diagnostics (Ramelow et al. 1993, Whitby et al. 1993, Süss et al. 1997, Schrader and Süss 1999, Wicki et al. 2000, Schwaiger and Cassinotti 2003, Rudenko et al. 2004, Tkachev et al. 2008). Moreover, real-time PCR methods utilizing biotin-labeled and exonuclease probes have been developed (Schwaiger and Cassinotti 2003, Achazi et al. 2011, Andreassen et al. 2012). A reverse-transcriptase loop-mediated isothermal amplification method that can differentiate TBEV subtypes and can be used for clinical diagnosis and surveillance has been optimized (Hayasaka et al. 2013). Detection of PCR products via hybridization probes and monoclonal antibodies to double-stranded DNA (Schreier et al. 1994) and multiplex detection of three TBEV subtypes (Ruzek et al. 2007) were also reported. In general, nested PCR assays usually provide enhanced specificity and sensitivity, albeit with an increased contamination risk. Real-time PCR is better suited for clinical specimens and high throughput screening, can provide quantitative results, and is less prone to contamination with a reduced turnaround time. In addition to the virus-specific PCR assays, TBEV sequences can further be identified via the protocols developed for generic detection of flaviviruses based on standard/nested or real-time PCR methods (Fulop et al. 1993, Kuno et al. 1998, Sanchez-Seco et al. 2005, Dyer et al. 2007, Moureau et al. 2007, Maher-Sturgess et al. 2008, Johnson et al. 2010, Vazquez et al. 2012). These protocols rely universally on sequencing of the products for characterization of different strains. Currently, comparative data on the performance of these assays are limited, including subtype sensitivity and detection limits for TBEV.

Table 2.

Examples of Primer and Probe Sequences of Nested PCR and Real-Time PCR for Detection of Tick-Borne Encephalitis Virus

	Name	Sequence	Position (nt)	Product size (bp) ^a	Target gene	Subtype specificity
Protocol by Puchhammer-Stöckl et al. 1995
First PCR	FSM-1	5′-GAG GCT GAA CAA CTG CAC GA-3′	7687–7706	357	NS5	European
	FSM-2	5′-GAA CAC GTC CAT TCC TGA TCT-3′	8024–8044
Second PCR	FSM-1i	5′-ACG GAA CGT GAC AAG GCT AG-3′	7748–7768	251
	FSM-2i	5′-GCT TGT TAC CAT CTT TGG AG-3′	7980–7999
Protocol by Achazi et al. 2011
	TBE F	5′-TGG AYT TYA GAC AGG AAY CAA CAC A-3′	2966–2990	98	NS1	European, Siberian, Far Eastern
	TBE R	5′-TCC AGA GAC TYT GRT CDG TGT GGA-3′	3041–3064
	TBE probe	5′-FAM-CCC ATC ACT CCW GTG TCA C-MGB-NFQ-3′	2996–3014

According to the tick-borne encephalitis virusstrain Neudoerfl, GenBank acc. no. U27495.

F, forward; NS, nonstructural protein; R, reverse; TBE, tick-borne encephalitis.

The shortcomings of the currently used TBEV PCR methods are that some of the protocols have been optimized for selected viral subtypes (often the European subtype), and considerable variation in the sensitivity and specificity is observed as revealed in an external validation study (Donoso-Mantke et al. 2007). For the assays capable of detecting all TBEV isolates, subtype determination relies on sequencing/pyrosequencing or amplicon size variation during electrophoresis (Ruzek et al. 2007, Achazi et al. 2011). Subtype determination via Sanger sequencing can be inefficient with less sequencing data, a disadvantage that can be overcome with pyrosequencing or reamplification of the initially positive samples via assays producing larger amplicons (Achazi et al. 2011, Andreassen et al. 2012) in cases where partial genomic characterization is required. Southern blotting and hybridization with specific probes can be performed (Ramelow et al. 1993, Puchhammer-Stockl et al. 1995, Schrader and Süss 1999), although it is impractical and time consuming for most applications. Alternatively, a combination of standard, nested, or real-time PCR assays with different genomic targets can be employed, an approach that enhances the detection performance and enables wider subgenomic characterization (Skarpaas et al. 2006, Katargina et al. 2013).

The diagnostic impact of TBEV NAT is limited to the viremic stage of the clinical disease in humans, usually before the onset of neurological symptoms and the emergence of specific immune responses (Holzmann 2003). Thus, the low prevalence of TBEV RNA detection for diagnosis in humans has been previously described in several reports. For example, only one serum and CSF specimen were TBEV RNA positive in a cohort of 46 sera and 59 CSF from patients with serologically confirmed TBE (Puchhammer-Stockl et al. 1995). Similarly, among 14 sera and 21 CSF samples from patients with clinical symptoms of TBE, a single CSF sample was reactive via RT-PCR (Schwaiger and Cassinotti 2003). In 454 blood samples from patients with different forms of TBE, i.e., from inapparent to meningeal with a two-wave clinical course, only 14 samples were PCR positive (Tkachev et al. 2008). The low TBEV RNA prevalence in patients could be due to the strict requirements for successful sampling combined with the instability of virions and viral RNA in the specimens and with the short viremia associated with only mild and unspecific clinical symptoms. However, NATs can be utilized in the early differential diagnosis of persons following a tick bite and/or during the febrile stage, as well as in individuals with undetectable antibodies and for post-mortem analyses of infected tissues (Tomazic et al. 1997, Schwaiger and Cassinotti 2003, Saksida et al. 2005). Recently, the presence of TBEV RNA in urine was reported in two of the four cases investigated during the encephalitic phase (Veje et al. 2014). Furthermore, shedding and RT-PCR detection of the virus in the urine over 6 weeks was described in a TBEV-infected individual with immunosuppression (Caracciolo et al. 2015). These observations suggest that urine sampling can represent a new diagnostic opportunity for NAT during the second phase; however, it requires revaluation in larger patient cohorts.

The optimal choice of NAT methodology depends on several factors involving the orientation (diagnosis and/or research), setting (total number of specimens received, infrastructure, personnel capabilities), and major goals (surveillance, detailed characterization etc.).

Serological Tests

Detection of TBEV antigens or virally induced antibodies via an antigen–antibody interaction constitutes the basis for serological testing. Prior to the dissemination of solid-phase assays, TBEV serological diagnosis relied upon hemagglutination inhibition, complement fixation, and neutralization assays that required the demonstration of a four-fold increase in antibody titers in sera from patients in the acute and convalescent phases (Holzmann 2003). These assays have mostly been replaced by rapid solid-phase assays; namely, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and immunoblotting. These tests provide results for specific antibody fractions (immunoglobulin G [IgG] or immunoglobulin M [IgM]), as well as increased sensitivity and specificity. Quantitative or semi-quantitative interpretation of data is also possible for certain assays. Crude antigens of the cell-culture-grown TBEV, recombinant prM/E proteins, and subviral particles are employed as test antigens in various assays (Sonnenberg et al. 2004, Obara et al. 2006). ELISA is currently the most commonly used technique and appears to be the method of choice for the serological diagnosis of TBE in several laboratories (Niedrig et al. 2007). The use of commercially available kits is recommended, and comparable results for immunofluorescence assays and ELISAs for IgG detection are reported (Litzba et al. 2014). Recent developments include ELISA and ELISA-based protein arrays that can simultaneously detect several flavivirus infections, including TBEV, with increased sensitivity compared with conventional ELISAs (Wang et al. 2015).

A solid-phase antigen immunoassay based on specific bioluminescent hybrid protein as a probe also provides enhanced detection of TBEV infections (Burakova et al. 2015).

Diagnosis of TBE in humans is based primarily on the demonstration of specific antibodies, which are usually detectable at the beginning of the second disease phase and that rapidly rise to high titers (Holzmann 2003, Gunther and Lindquist 2005). Serum or CSF can be tested for anti-TBEV IgM and IgG antibodies. At the beginning of the neurological phase, sera from more than 90% of patients have the specific IgM antibodies and frequently also IgG antibodies. If the antibodies are absent or of low titer, appearing as an indeterminate result, collection and reanalysis of a new sample after a minimum period of one week is recommended. At the beginning of the neurological phase of TBE, anti-TBEV antibodies are rarely present in CSF. Detectable levels of intrathecal antibodies during TBE are usually observed between 10 and 14 days after the advent of neurological symptoms. In case of increased permeability of the blood–brain barrier, the antibodies can, however, also passively cross from the blood to the CSF at earlier stages of the neurological phase (Günther et al. 1997). Investigation of anti-TBEV antibodies in CSF therefore provides only limited information, and precise interpretation may require a demonstration of intrathecal synthesis calculated according to the Reiber method (Reiber and Lange 1991). Commercially available standardized assays for TBEV antibodies in blood-CSF pairs are marketed.

Several IgM and/or IgG profiles can be observed during TBEV serological testing while evaluating a clinically suspected case as follows:

Specific IgM and IgG negative (Ig total negative): This can represent a patient with a central nervous system infection other than TBE or a patient at a very early stage of the infection when the antibodies are not present. Testing of a control sample after at least a one-week interval is necessary. TBEV-specific or generic flavivirus NAT should be considered in available samples.

Specific IgM positive, IgG negative: This profile represents a suspected TBE case at an early stage of the infection. Testing a control sample after at least a one-week interval is recommended. IgM-based serodiagnosis may be hampered by IgM persistence due to previous exposure unrelated to the current disease or vaccination, as well as the detection of cross-reactive antibodies due to flaviviral infections other than TBEV. Quantitation of IgM provides important information in such instances, where a recent TBEV infection is characterized by antibody levels above 500 arbitrary units (Stiasny et al. 2012).

Specific IgM positive, IgG positive: This profile represents a proven TBE case, given that the antibody specificity is confirmed. However, similar results can also be seen in patients who received a recent TBEV vaccination (first or second vaccination within the previous months) or, rarely, in cases with a persistent IgM response. In case of any indeterminate results, a follow-up sample can be tested for the determination of four-fold antibody titer increase or IgG avidity testing can be employed (Table 3).

Table 3.

Interpretation of the TBE Virus Assay Results in Circulation of Clinically Compatible Individuals

Assay
NAT	IgM	IgG	Interpretation ^a
−	−	−	No TBE—test for other agents
			Improper sampling and storage—request new sample
			Suboptimal diagnostic assays—perform internal/external quality controls
+	−	−	Early TBE (before seroconversion), recent exposure
+	+	−	Probable TBE (recent seroconversion)—test for IgM specificity, request follow-up sample
−	+	−	Probable TBE—test for IgM specificity, request follow-up sample
−	+	+	Probable TBE —test for antibody specificity, IgG avidity, CSF antibodies
+	+	+	Improbable result —retest for NAT, confirm antibody specificity

Recommended follow-up tests may differ according to the institutions and local guidelines.

NAT, nucleic acid tests.

Avidity determination of IgG provides additional information for the serological diagnosis of TBEV infection. Increasing avidity results in increasing stability of the antigen–antibody complex in the presence of dissociating agents, (usually urea). Treatment of antigen–antibody complexes for 3 min with 7 M urea can differentiate well between high-avidity antibodies, which are not removed, and low-avidity antibodies, which are removed from the antigen. This allows differentiation between low-avidity IgG present during early stages of TBE and high-avidity IgG produced at later stages of the infection or persistent IgG after previous exposure. The major advantage of this assay is in the detection of acute cases of TBE without measurable IgM in the available serum samples (Gassmann and Bauer 1997). Several commercial TBEV IgG ELISA kits are also available for avidity determination.

Another widespread and well-known problem of diagnostic TBEV serology is the antibody cross-reactivity among flaviviruses (Allwinn et al 2002). A previous flavivirus exposure or vaccination, most notably with West Nile, Japanese encephalitis, and dengue or yellow fever viruses, is likely to produce false positive results in the aforementioned serological assays (Niedrig et al. 2007). Given that some of these infections may also present with symptoms involving the central nervous system, a specific diagnosis of TBEV may be seriously hampered by the potential cross-reactions, especially during late stages of the disease where serology remains the sole diagnostic method (Haglund et al. 2003). Although significantly improved specificity values have been reported in some current assays (Sonnenberg et al. 2004, Wang et al. 2015), the most reliable confirmatory method to rule out false positivity in TBEV diagnostic serology is the VNT (Tables 1 and 4). However, current regulations in a number of Western countries restrict the performance of this assay to laboratories equipped with a biosafety level 3 facility; thus it is rarely requested and performed during routine clinical diagnostics (Hierholzer and Killington 1996, Holzmann 2003). Practical alternatives to conventional VNT are based on fluorescent focus inhibition (Vene et al. 1998) or virus-like particles expressing a reporter gene, instead of the live virus strains (Yoshii et al. 2009) (Table 4).

Table 4.

Examples of Virus Neutralization Test Protocols Employed for TBE Virus

VNT by Vene et al. 1998; Donoso-Mantke et al. 2011
1.	Dilute sera 1:5 in Eagle's MEM (+3% inactivated fetal calf serum; MEM-FCS)
2.	Inactivate the diluted sera at 56°C for 30 min
3.	Prepare two-fold serial dilutions in MEM-FCS using 50 μL aliquots in 96-well culture plates
4.	Add TBEV at approximately 50 FFD₅₀ in 50 μL per well
5.	Incubate the plates at 37°C and 5% CO₂ for 90 min. Then add 100 μL of cell supension (5 × 10⁵ BHK-21 per mL in MEM-FCS)
6.	Incubate the plates for 24 h at 37°C and 5% CO₂
7.	Wash the cells with PBS, add 200 μL cold acetone
8.	After 1 h at −20°C, discard acetone and air dry plates for 30 min at RT
9.	Add 75 μL of rabbit anti-TBEV serum to visualize the virus foci
10.	After 45 min incubation at 37°C, wash the plates three times with PBS, add 75 μL of conjugate, incubate 45 min
11.	Wash the plates three times with PBS, examine 20 microscopy fields per well for fluorescent foci
12.	Calculate neutralizing antibody titers are the reciprocal of the serum dilution that reduce the challenge virus to one FFD₅₀

VNT by Bárdoš et al. 1983, modified by Palus et al. 2013
1.	Dilute sera 1:4 in Leibowitz L-15 medium supplemented with 1% antibiotics (penicillin, streptomycin, amphotericin B) and 3% foetal bovine serum
2.	Inactivate the diluted sera at 56°C for 30 min
3.	Prepare two-fold serial dilutions in L-15 medium using 50 μL aliquots in 96-well culture plates
4.	Add 1 × 10³ of TBEV strain Hypr (the virus dose was adjusted to cause almost confluent plaques with 90–95% cytolysis) in 50 μL per well
5.	Incubate the plates at 37°C for 90 min, then add 100 μL of cell suspension of porcine kidney stable (PS) cells (5 × 10⁴ PS cells per well)
6.	Incubate the plates for 4–6 days at 37°C
7.	Remove the cell supernatant, wash the cells with PBS, fix and stain the cells by 0.1% naphthol blue black in 6% (v/v) acetic acid for 45 min
8.	The highest serum dilution that caused a 90% reduction of plaques is regarded as the endpoint titer

BHK, baby hamster kidney cells; FFD50, 50% focus forming dose; MEM, minimum essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; RT, room temperature; TBEV, TBE virus.

Similarly to human clinical samples, serological detection of TBEV infection in rodents and other vertebrates is based mainly on ELISA and VNT. Commercial all-species or species-specific ELISAs can be used to detect anti-TBEV IgG antibodies in milk or sera from a large variety of susceptible animals (Cisak et al. 2010, Ikawa-Yoshida et al. 2011, Klaus et al. 2011, Širmarová et al. 2014). It is reported that nonspecific ELISA reactivity varied among animal species and age of the animals tested and that VNT is required for specificity confirmation (Klaus et al. 2011).

It should be stated that surveillance strategies, case definitions, and diagnostic laboratory criteria vary among European Union countries with endemic occurrence of TBE. Overall, for the evaluation of suspected human cases, NAT is likely to be of significance in the early stages of TBEV-associated diseases when there is sufficient number of virions in affected tissues or in circulation, whereas serology is likely to be the mainstay of diagnosis in later stages after the induction of specific immune response.

Conclusions and Future Perspectives

The diagnosis of TBEV infections in humans still presents certain problems due to variations in clinical presentation and optimal assays suitable for different infection stages. External quality control programs revealed an ongoing need for improvement in sensitivity/specificity of the widely used NAT and serological assays, as well as the impact of quality control measures (Donoso-Mantke et al. 2007, Niedrig et al. 2007). Development toward newer assays with reduced cross-reactivity to heterologous flaviviruses improves specificity, where antigen detection assays and quantitation of IgM appears promising. Moreover, assays that can reliably differentiate vaccinations and previous exposure from recent infections likely to be associated with clinical symptoms are required. Sensitive and standardized NAT assays can significantly enhance virus detection during the early stages of infection after exposure, and may support serological diagnostics before/during seroconversion. Rapid and sensitive antigen/antibody assays as well as isothermal amplification methods for NAT detection will facilitate field surveillance as well as the preliminary diagnosis in primary care centers. Investigation of immune responses in TBEV infections are instrumental in identifying key epitopes for antibody detection (Jarmer et al. 2014). Currently available assays and testing algorithms must be overviewed according to specific needs and local epidemiological features, and updated guidelines should be provided by public health organizations. Considering the expansion of the TBEV-affected areas and increases in travel, TBEV remains an important etiological agent in the workup of cases with compatible clinical presentation (Haditsch and Kunze 2013).

Footnotes

Acknowledgments

This study was supported by the Russian Scientific Foundation (project No. 14-15-00615); the Academy of Sciences of the Czech Republic (Z60220518), Czech Science Foundation project Nos. P502/11/2116 and GA14-29256S, and the project LO1218, with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the NPU I program. The work of KE and DR was done under the frame of EurNegVec European Cooperation in Science and Technology Action TD1303. KE is a recipient of the Georg Forster Research Fellowship (HERMES) for Experienced Researchers Award of the Alexander Von Humboldt Foundation, 2015.

Author Disclosure Statement

No competing financial interests exist.

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