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Table of Contents
Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 212-220

Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus

ICMR–National Institute of Virology, Pune, India

Date of Submission15-Feb-2018
Date of Acceptance25-Jun-2018
Date of Web Publication09-Jul-2020

Correspondence Address:
Dr Devendra Mourya
ICMR–National Institute of Virology, Sus Road, Pashan, Pune–410 021, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.289392

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Background & objectives: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available.
Methods: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions.
Results: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison.
Interpretation & conclusion: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus.

Keywords: Bacterial expression; ELISA; Kyasanur forest disease; non-structural protein1

How to cite this article:
Yadav P, Chaubal G, Jena S, Shil P, Mourya D. Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus. J Vector Borne Dis 2019;56:212-20

How to cite this URL:
Yadav P, Chaubal G, Jena S, Shil P, Mourya D. Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus. J Vector Borne Dis [serial online] 2019 [cited 2023 Mar 30];56:212-20. Available from: http://www.jvbd.org//text.asp?2019/56/3/212/289392

  Introduction Top

Since the identification of Kyasanur forest disease (KFD) in 1957[1], there have been an estimated 400–500 cases recorded per year in India[2],[3]. The KFD virus (KFDV) is maintained by ticks, mammals, and bird cycles. The black-faced langur (Presbytis entellus) and the red-faced bonnet monkey (Macaca radiata) act as sentinel animals, because they are as susceptible to KFDV, as humans[4],[5]. Epizootics of this infection cause a heavy burden of mortality in these monkeys, which indicates likely outbreaks, mostly from December to June. More or less engorged ticks drop off as soon as the animal dies, thus generating hotspots for infection[6].

Rapid diagnostic methods based on nested reverse transcriptase-polymerase chain reaction (RT-PCR), realtime RT-PCR and immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) have been described earlier[7]. Recent reports of emergence of KFD in newer areas like Bandipur Tiger reserve[8], Malappuram district of Kerala State[9] and Sindhudurg district of Maharashtra state[10] are of concern. Presence of this virus in a human patient has also been reported in China, close to the Indian border[11]. These reports serve as a reminder that KFD is a significant public health problem and is spreading to previously uninfected regions like Sindhudurg district, Maharashtra state.

As seen in the case of dengue virus (DENV), a mosquito-borne flavivirus, non-structural protein 1 (NS1) of KFDV may be a potential biomarker for early diagnosis of disease, and kits for the same are commercially available[12],[13]. Studies have also been conducted using the NS1 protein of West Nile virus (WNV)[14],[15], Japanese encephalitis virus (JEV)[16],[17], Murray valley encephalitis virus (MVEV)[18] and tick-borne encephalitis virus (TBEV)[19],[20]. To date, there has been no report for the development of recombinant NS1 protein–based antigen capture ELISA for early diagnosis of KFD.

In the present study, we assessed, whether the KFDV NS1 protein could be used as a potential biomarker for early detection of the disease through the development of an antigen capture ELISA using bacterially expressed recombinant KFDV NS1 protein.

  Material & Methods Top

Amplification, cloning, expression and purification of KFDV NS1 protein

Primers for full length NS1 gene were designed using KFDV P9605 strain sequence (GeneBank accession No: JF416958.1). The NS1 gene (Size: 1062 bases, position: 2463–3524) encodes for a 354 amino acid protein. Primers used for amplification were: GCA TGG ATC CGA TAT GGG CTG TGC A (KFDV NS1 forward primer) and ATA TGC GGC CGC ATC AGC CAG CAC CAT C (KFDV NS1 reverse primer). PCR amplification was undertaken using superscript III one-step RT-PCR system with platinum Taq high fidelity kit (Invitrogen, Life Technologies, USA). A 1062 bp KFDV NS1 gene was obtained, purified (Qiagen gel purification kit) and confirmed by sequencing using Sangers sequencing method by ABI Dye kit (Applied Biosystems) the primers used for amplification were also used for sequencing. BamH I and Not I restriction sites were incorporated in the forward and reverse primer, respectively for cloning, performed in DH5-alpha cells (Invitrogen, Life Technologies, USA), the KFDV NS1 gene in pET28a vector (Invitrogen, Life technologies, USA). The confirmed clone was transformed in BL21 (DE3) cells (Invitrogen) and further confirmed by restriction digestion and sequencing. Induction of the bacterial cells for expression of KFDV NS1 protein was standardized at 1mM IPTG at 37 °C for 3 h.

A C-terminal histidine tag was present on the translated protein as the sequence for poly-histidine is present on pET28a vector. The expressed protein was, therefore purified using Probond Nickel chelating resin. The induced cell pellet was resuspended in native conditions and lysed in the presence of Triton X-100 and lysozyme. After binding to the nickel column, the protein was eluted using varying concentrations of imidazole. The nickel column was washed with 10mM and 20mM imidazole containing buffer after binding of protein lysate. The protein was eluted in 100mM fractions. The purified protein was validated against KFDV positive human serum in western blot and was found to react specifically with KFDV positive serum. We used KFDV real-time rNApositive, IgM and IgG positive serum samples for standardization of western blot.

Development of KFDV NS1 antigen capture ELISA

Six to eight weeks old BALB/c mice were immunized with 5μg purified recombinant NS1 protein (in sterile PBS) in Freund’s adjuvant and used for immunization. Four control mice were immunized with sterile PBS in Freund’s adjuvant using routine immunization protocol. This anti-NS1 mouse serum was used for coating ELISA wells. Further, six to eight weeks old New Zealand white rabbits were immunized with 100 μg purified recombinant NS1 protein (in sterile PBS) in Freund’s adjuvant and used for immunization using routine immunization. one control rabbit was immunized with sterile PBS in Freund’s adjuvant. The anti-NS1 rabbit serum was purified, biotinylated and used as the detection antibody in ELISA. Briefly, anti KFDV-NS1 mouse polyclonal serum was coated onto ELISA wells (Nunc, MaxiSorp strips) at a dilution of 1:10 in carbonate-bicarbonate buffer (pH 9.2, 0.05 M) and incubated overnight at 4 °C. The wells were blocked with liquid plate sealant (Candor Bioscience, Germany) at 37 °C for 2 h. Human serum samples (dilution 1:1) in sample diluent (PBS+1% BSA) were dispensed into coated and blocked wells and incubated at 37 °C for 1 h. Recombinant KFDV NS1 protein was used as positive control. The wells were washed five times with wash buffer {10mM PBS pH 7.4 with 0.1% Tween-20 (Sigma, USA)} and purified and biotin conjugated anti- KFDV NS1 antibodies raised in rabbit were dispensed at a dilution of 1:50 and were incubated at 37 °C for 1 h. The wells were washed five times with wash buffer and streptavidin-HRP (Sigma, USA) was dispensed into each well at a dilution of 1:5000 and incubated at 37 °C for 30 min followed by washing five times with wash buffer. Tetramethyl benzidine (Clinical Science, USA) substrate was added on to the plate and incubated in dark for 15 min at room temperature. The reaction was stopped by adding 1 N sulphuric acid. The wells were read at 450 nm using appropriate ELISA plate reader. Appropriate controls were included in the test. All work with KFDV positive samples were handled in BSL2 laboratory following in-activation.

Testing specificity and sensitivity of KFDV NS1 antigen capture ELISA

To exclude the possibility of the cross-reactivity with other flaviviruses and to test specificity, known positive serum samples of JEV, WNV, and DENV were incorporated during standardization as an NS1 antigen capture ELISA available commercially for dengue. A total of 25 dengue NS1 positive samples were tested. Human samples positive for Crimean-Congo hemorrhagic fever virus (CCH-FV) and Nipah virus were also used as outliers. Several negative samples (serum samples from healthy volunteers of different age groups from both sexes who had no history of KFDV infection) were also included in the test.

To test the sensitivity of the assay, varying concentrations of KFDV NS1 protein (from 50 (μ/well to 1 (μ/well) were used in the assay. NS1 protein was diluted either in PBS or in spiked KFDV negative serum sample. Spiking negative serum with KFDV NS1 protein is believed to assist in determining whether the assay is inhibited by proteases or any other inhibitors present in human serum and may thus mimic the actual conditions while testing samples.

In silico analysis

Predicting protein half-life, instability index: The protein half-life and instability index for NS1 proteins of KFDV, TBEV, DENV, JEV and WNV were compared theoretically using ExPASy ProtParam tool (http://web. expasy. org/protparam/protparam-doc.html).

3D structure, B-cell epitope and glycosylation prediction and comparison

The NS1 amino acid sequence of KFDV was compared with that of DENV and TBEV using the online FAS-TA software fasta/virginia.edu). As NS1–based antigen capture ELISA is commercially available for DENV, a mosquito-borne virus, and TBEV; and KFDV share a common host and are closely related flaviviruses, these two viruses were considered for comparison with KFDV. The 3D structures for the NS1 protein from KFDV and TBEV were predicted using the homology-based modeling protocol as implemented in the SWISS-MODEL online workstation in automated mode. The predicted models were evaluated by PROCHECK analyses (www.ebi. ac.uk/). Minimized energy for the structures was obtained using Swiss PDB Viewer (SPDBV) tool and rendering of images was carried out in Discovery Studio 3.1. B-cell epitopes were predicted for the amino acid sequences of NS1 from DENV, KFDV and TBEV using the Kolaskar method for antigenic predictions as implemented in the online services of Immune Epitope Database and Analyses Resource (IEDB; www.iedb.org/). The N-linked glycosylation sites were predicted from protein sequences using the NetNGlyc webserver (www.cbs.dtu.dk/services/NetNGlyc/).

Ethical statement

The Indian Council of Medical Research–National Institute of Virology (ICMR–NIV) Pune has been involved in the investigations of viral disease outbreaks of human including zoonosis in India. The Institute has a policy to obtain approval on the projects which involves animals from the national committee called ‘‘Committee for the Purpose of Control & Supervision of Experiments on Animals (CPCSEA)” under the Ministry of Environment and Forests, Government of India. Our study project number MCL1405/HEP1315 was approved by the Institutional Animal Ethical Committee (IAEC) permitting the use of adult mice and rabbits as laboratory animals for the development of antibodies, respectively. Institutional Human Ethical Committee, ICMR–NIV Pune was informed where the human serum samples were used during KFD outbreaks (project No.—NIV/HEC/2016/D-320). All the study participants provided informed consent. The consent was in written format, both in English and local language (Marathi). All the records analyzed were anonymized. Every sample was registered in the central registry of the Institute and allotted a specific number, which was used throughout the study.

  Results Top

Amplification, cloning, expression and purification of KFDV NS1 protein

The confirmed 1062 bp PCR product was cloned in pET28a vector and expressed in BL21 cells. The expression of protein was confirmed by SDS PAGE [Figure 1]a and western blotting [Figure 1]b and [Figure 1]c with KFDV positive human serum and anti-histidine antibody (Sigma, UK), wherein a 43 kDa band was detected. The partially purified protein was used to raise immune sera in mice and rabbits.
Figure 1: (a) Confirmation of KFD NS1 protein expression by Coomassie staining of 12.5% SDS PAGE. Lane 1: Lysate of uninduced BL21 (DE3) cells; Lane 2: KFD NS1 protein expressed in BL21 (DE3) cells after induction with 1mM IPTG at 37°C for 4 h; Lanes 3, 4, 5: Elution fractions 1, 2 and 3 of induced cell lysate purified using Nickle chelating column; and Lane 6: Molecular weight marker; (b): Confirmation of KFD NS1 protein expression by Western Blot using commercially available anti-histidine monoclonal antibody. Lane 1: Protein lysate before purification showing reactivity with anti-histidine antibody; Lane 2: KFD NS1 protein showing reactivity at expected size (42 kDa) with anti-histidine monoclonal antibody; and (c) Confirmation of KFD NS1 protein expression by Western Blot using human serum sample positive for anti-KFD IgM and IgG antibodies, thereby increasing the probability of the presence of anti-KFD NS1 antibodies. Lane 1: Protein lysate before purification showing reactivity with KFD positive human serum; Lane 2: KFD NS1 protein showing reactivity at expected size (42kDa) with KFD positive human serum.

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Testing specificity and sensitivity of KFDV NS1 antigen capture ELISA

The assay specifically detected KFDV NS1 antigen and was negative for JEV, WNV, DENV, CCHFV and Nipah virus. In the case of DENV, as this is the only virus against which an NS1 antigen capture ELISA is commercially available, 25 DENV NS1 positive samples were tested and found to be negative for KFDV NS1. In terms of sensitivity, the antigen capture ELISA could detect up to 1μg recombinant NS1 protein both when it was diluted in PBS and when the protein was used to spike KFDV negative serum. The OD 492 values for the antigen capture ELISA ranged from 1.1 for 50 μg NS1 protein to 0.18 for 1 μg NS1 protein. An OD of 0.12 was considered as cut-off (based on the mean of three negative controls and adding 3 times the standard deviation to this value).

Testing samples with KFDV NS1 antigen capture ELISA

A total of 130 samples were tested. Based on the real time-PCR, anti-KFDV IgM ELISA and anti-KFDV IgG ELISA results, samples were divided into eight groups. The results are detailed in [Table 1]. Out of 43 samples that were found to be indicative of early stage of infection, only two samples were positive for KFDV NS1 antigen. One sample out of 32 negative samples showed positivity of KFDV NS1 antigen. All the other groups tested negative. The data are clearly indicative that NS1 antigen may not be a diagnostic marker in case of KFDV infection.
Table 1: Human serum samples tested using KFDV NS1 antigen capture ELISA

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In silico analysis

Predicting protein half-life, instability index: Based on the calculations, as obtained from ProtParam software tool, it was seen that all DENV, TBEV, JEV, WNV and KFDV have a predicted in vivo half-life of 1.1 h. However, the instability index for KFDV was higher (52.60) when compared to DENV (varying from 35.07 to 45.27 for the four subtypes), JEV (32.7), TBEV (45.01) and WNV virus (46.42), thus indicating that the KFDV NS1 protein is unstable in comparison [Table 2].
Table 2: Comparison of protein half-life, instability index and aliphatic index calculation

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3D structure, B-cell epitope andglycosylationprediction and comparison: The 3D structures for NS1 protein (amino acid 1-354) from KFDV was obtained by homology modeling using DENV NS1 protein (4o6B.pdb) as a template (target-template identity = 40 %). PROCHECK analyses revealed that for KFDV NS1, the occupancy of Ramachandran plot was 99% (non-glycine amino acids in most favoured + additionally allowed + generously allowed regions). The 3D structure of TBEV NS1 was obtained using WNV NS1 as template (4TPL.pdb), with the target-template identity of 43.7 %. The occupancy of Ramachandran plot was 98.3 % (non-glycine amino acids in most favoured + additionally allowed + generously allowed regions). The minimized energy of the KFDV NS1 as obtained from SPDBV was –13465.20 kJ/mol and comparable to that of the template (–14312.79 kJ/mol). The minimized energy of the TBEV NS1 was –12388.26 kJ/mol, and comparable to that of the template 4TPL.pdb (–18061.6 kJ/mol).

[Figure 2] shows the 3D folds of NS1 proteins from DENV, KFDV and TBEV along with the surface electrostatics. Though folds were similar [Figure 2]a, considerable differences exist locally between the proteins in surface contour and surface electrostatics due to differences in amino acid composition (amino acid identities are in the range fo 50–75%) [Figure 2]b. The NS1 172-352 (NS1 fragment amino acids: 172-352) homodimers from dengue, KFDV and TBEV have been compared [Figure 3]. The beta-ladder surface is known to form the interface between subunits in the dengue NS1 hexamer, whereas the loop surface of the NS1 172-352 [Figure 3]a forms the part of the hydrophobic cavity that helps in the formation of a lipoprotein complex, which is the final functional conformation[27]. Considerable variations exist between flaviviruses in the loop surface of NS1 172-352, which determines interactions with host factors [Figure 3]b.
Figure 2: (a) Fold of the NS1 protein from dengue virus (DENV NS1), Kyasanur forest disease virus (KFDV NS1) and tick-borne encephalitis virus (TBEV NS1); and (b) Surface electrostatics for the proteins as obtained from NOCH software.

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Figure 3: Comparison of the NS1172–352 (NS1 fragment amino acids: 172–352) homodimers between dengue, KFDV and TBEV–(a) 3D folds and surface electrostatics of the β-ladder surface; (b) 3D folds and surface electrostatics of the loop surface (rotated 180° from β-ladder surface).

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Superimposition of the monomers of KFDV NS1 and DENV NS1 revealed a root mean square deviation (RMSD) of ~0.91 Å covering 1200 backbone atoms. This implies that the structures are similar in terms of 3D fold. However, the identity of amino acid composition between the two proteins is 37.6% and similarity being 65.5%. This implies that there are ~34% of amino acids which are complete mismatch in terms of properties. In terms of structure, these may contribute to minor changes in the folds, but will alter the surface contour and electrostatics locally. Such alterations may affect antigenicity, protein function and interactions with other biomolecules. Superimposition of KFDV NS1 and TBEV NS1 revealed a RMSD ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins is 73.4% and similarity being 92.9%, as revealed from the pairwise comparison.

Results of B-cell epitope predictions based on Kolaskar method for the DENV, KFDV and TBEV NS1 are summarized in [Table 3]. Some of the predicted epitopes are similar, but majority demonstrate differences between DENV and KFDV. There is greater similarity between epitopes from KFDV and TBEV. N-glycosylation sites on DENV type 2 are located at: 130-NQFT-133 and 207-NDTW-210. Similarly, those for KFDV are located at 85-NLTV-88, 208-NATG-211 and 224-NCTW-227 and for TBEV at 84-NLTV-87, 203-NDTG-206, and 219-NCSW-222. Comparison among DENV, KFDV and TBEV revealed that two of the N-glycosylation sites of KFDV occur on the exposed B-cell epitopes: (i) 208- NATG-211 on epitope 210-TGVFISELIVTD-221; and (ii) 85-NLTV-88 on epitope 84-ANLTVVVD-91 [Figure 4] and [Figure 5]. This indicates that glycosylation may play a role in the antigenicity and immunogenicity of the protein when used for immunization in rabbits and mice. Hence, expression of KFDV NS1 protein in a bacterial system (where glycosylation does not occur) for the development of antigen capture ELISA would not be ideal. In contrast, the glycosylation sites of Dengue NS1 protein were not present in or near to any of the B-cell epitopes. Hence, Dengue NS1 based antigen capture ELISA can be developed in a bacterial system, as the epitopes are not masked by glycosylation.
Table 3: Predicted B-cell epitopes based on Kolaskar antigenicity.

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Figure 4: N-glycosylation sites marked on predicted KFDV NS1 structure: the KFDV NS1 protein fold shown in Blue. Exposed epitopes (orange) with N-glycosylation sites indicated in space fill mode with Green colour.

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Figure 5: Multiple sequence alignment of the NS1 protein from dengue (DENV2 NS1), Kyasanur forest disease (KFDV NS1) and tick-borne encephalitis (TBE NS1) viruses showing N-glycosylation sites

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  Discussion Top

A potential driving role of DENV NS1 protein in disease pathogenesis has been previously suggested following the observed association between high levels of complement consumption and the more severe form of dengue disease—dengue shock syndrome (DSS)[22],[23]. It appears that the levels of secreted NS1 differ significantly between flaviviruses, although there are limited reliable in vivo data to allow accurate soluble NS1 quantification except for DENV. The levels of soluble NS1 in DENV are substantial, with levels in excess of 10 mg/ml reported[24]. This led to the development of an early diagnostic NS1 antigen capture assay for DENV. However, no reliable data are available on the level of NS1 antigen secreted in the case of KFDV.

Moreover, although DENV and KFDV are both vector-borne flaviviruses, the mode of transmission is different. DENV is transmitted by Aedes mosquitoes and causes high-grade fever lasting for 3 days to 1 week, severe headache (mainly retrobulbar), lassitude, myalgia and painful joint, metallic taste, appetite loss, diarrhea, vomiting, and stomach ache[25]. The KFDV is tick-borne and is characterized by chills, a frontal headache, body aches, and a high fever for 5–12 days; the case-fatality rate[6] is 3–5%. A difference in the vector for transmission of these diseases may lead to difference in level of NS1 secretion and function.

In the absence of experimentally known 3D structure for KFDV NS1 protein, we used bioinformatics techniques of homology based modeling to obtain a 3D model structure. This was compared to the 3D structures of NS1 from: (i) DENV; and (ii) TBEV for which presence of neutralizing anti-NS1 antibodies has been documented[26]. Although the 3D fold of the backbone for all the three proteins is similar (RMSD range: 0.79–0.91 Å), differences exist in amino acid composition. Since the amino acid identity between DENV and KFDV is only 37.6%, a large number of differences exist across various domains of the protein, which may result in altered physico-chemical properties and surface electrostatics. This results in altered function especially interactions with other protein or host factors. This correlates with the recent observations by Song et al[27] who inferred that different flavivi- ruses have different NS1 characteristics.

The in silico Kolaskar method of b-cell epitope prediction[28] used in the study is reliable as it had correctly predicted B-cell epitopes in JEV E-proteins which were identified by experiments[29].

It is established that different flaviviruses have N-linked glycosyl sites at different positions in NS1 proteins and these differences may reflect variations in immune evasion strategy[22],[30],[31]. The glycosylation site N207 (DENV-2 sequence numbering) is common to all flaviviruses and plays important role in NS1 oligomer assembly and secretion[22],[32],[33],[34] as revealed from glycosylation abolishing experiments.

In dengue NS1, N207 occurs outside the experimentally known B-cell epitope 193-AVHADMGYWIES-204 and N130 is near but outside of known B cell epitope 112-KYSWKSWGKAK-123. Hence, glycosylation modification at these sites may not have any impact on antigenicity or interaction with host antibodies. The glycosylation sites in KFDV NS1 are: a) 208-NATG-211 on predicted epitope 210-TGVFISELIVTD-221 and b) 85- NLTV-58 on epitope 84-ANLTVVVD-91. Hence, these sites may play a role in antigenicity and immunogenicity of the KFDV NS1 when used for immunization in rabbit and mice. Consequently, the expression of KFDV NS1 in a bacterial system (where glycosylation modification does not occur) may not be ideal for the development of antigen capture ELISA.

Bioinformatics analyses in the study showed that N-glycosylation sites do occur at or in close proximity of exposed B-cell epitopes on KFDV NS1 protein indicating the possibility of glycosylation-induced masking of these epitopes. This is in contrast to that in DENV NS1. In silico stability analyses based on amino acid composition revealed that the KFDV NS1 is least stable of all the other tested flaviviruses.

The experimental results showed that even if an antigen capture ELISA could be developed for KFDV using bacterially expressed NS1 protein, it could not detect NS1 antigen in infected human serum samples. Screening of samples covering (i) early; (ii) intermediate; and (iii) late disease stages using the said ELISA demonstrated a weak positive response for NS1 protein in only two out of 43 early stage samples, while all other samples were found to be negative (n = 87). This is in contrast to the performance of the commercially available DENV NS1 ELISA kit. The contrast may be attributed to the difference in characteristics of NS1 protein from DENV and KFDV. In particular, the NS1 used in the developed ELISA was expressed in BL21 (DE3) E. coli, which does not permit glycosylation. Hence, the bacterially expressed KFDV NS1 may behave differently from the naturally expressed NS1 (human host) or NS1 expressed in eukaryotic systems viz. Baculovirus, mammalian cell lines or yeast, which are conducive to glycosylation. Since a commercial kit using bacterially expressed NS1 was available for DENV, we initially choose to work on bacterially expressed KFDV NS1.

  Conclusion Top

Considering the differences in characteristics (both structural and functional) between DENV and KFDV NS1 protein, it can be inferred that bacterially expressed NS1 protein may not be suitable for development of antigen capture assay for KFDV.

Conflict of interest: The authors declare that there is no conflict of interest.

  Acknowledgements Top

We would like to acknowledge the valuable contribution of staff of Maximum Containment Laboratory, NIV, Pune. In particular, we would like to thank Dr. Sachin Badole and Dr. Vimal Kumar for conducting raising immune sera, and Mr. Pravin Kore and Ms. Trupti Rale for conducting protein expression and purification experiments. We would also like to acknowledge Dr. Sarah Cherian, Head of Bioinformatics section, NIV, Pune for her critical reviewing of the manuscript and suggesting changes in the sequence analysis.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]

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