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West Nile virus (WNV), the etiologic agent responsible for West Nile encephalitis (3, 21), belongs to the genus Flavivirus. Besides WNV, many other flaviviruses also cause significant human diseases, including yellow fever virus (YFV), four-serotypes of dengue virus (DENV), Japanese encephalitis virus, and tick-borne encephalitis virus (4, 19). More than 50 million, 200 000, and 50 000 humans are infected by DENV, YFV and JEV every year, respectively (9). Flavivirus virions are about 50 nm in diameter, and harbor a plus-sense, single-stranded RNA genome of about 11 kb. The single open reading frame of the genome encodes a polyprotein, which is processed by viral and cellular proteases into three structural proteins [capsid (C), premembrane or membrane (prM/M), and envelope (E)] and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (5). Among those, the structural proteins are involved in viral particle formation. The NS proteins are primarily responsible for RNA replication; they also function in viral assembly (13, 15) and evasion of host immune response (16, 20). Serological assay is the dominant method for diagnosis of flavivirus infections in human. This is because, by the time patients develop symptoms and present to clinicians, immune response has already cleared the viruses or reduced the viremia to very low or undetectable levels. Upon flavivirus infection, majority of the neutralizing antibodies are directed against the E protein, although some antibodies may recognize the prM/M protein (6, 8, 22, 27). These antibodies are cross-reactive with other flavivirus members, leading to specificity as the confounding challenge of the serological diagnosis of flavivirus infections. The current serology assays use viral structural proteins (inactivated virus or recombinant E protein) as antigens in ELISA or Luminex formats (10, 14, 18, 26). These serology assays lack specificity, verified by cross-species plaque reduction neutralization tests (PRNT) before a specific virus of infection could be determined. PRNT is performed by testing the neutralization of a panel of flaviviruses by a potential flavivirus-infected serum (17); it represents the most accurate serologic method for differentiating infections of closely related flaviviruses. Since PRNT uses live viruses, the assay should be performed in biosafety level-3 or level-4 containment for many flaviviruses. Additionally, PRNT is time-consuming, requiring 10, 7, and 5 days to test DENV-1, YFV, and WNV, respectively (23). Here we describe a novel approach for virus-type specific diagnosis of flavivirus infection using viral-like particles (VLPs). The VLPs contains a luciferase-reporting replicon that is encapsidated and enveloped by the complete viral structural proteins. The VLPs have physiochemical and antigenic properties similar to the authentic virus particles, and can infect susceptible cells. However, the infection is a single-round, because no structural proteins are expressed upon the VLP infection. Using a WNV-infected mouse serum, we showed that the serum neutralized WNV VLP more selectively than the DENV-1 and YFV VLPs. The results demonstrate that VLPs can replace raw viruses for neutralization assays to differentiate closely related flaviviruses. Compared with the raw virus-based PRNT, the VLP-based neutralization assay is rapid ( < 1 day) and quantitative (measured by luciferase activity), and can be performed in biosafety level-2 laboratory.
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Vero and BHK-21 cells were maintained in Dulbecco modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37℃. WNV strain 3356 was used in this study. WNV-infected mouse serum (immune IgG) was a generous gift from Kristen Bernard (Wadsworth Center, Albany, New York). The anti-actin antibody was purchased from Sigma. The monoclonal antibody against WNV NS5 was in-house generated.
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Ten micrograms of flavivirus replicon containing a Rennila luciferase and the foot-and-mouth-disease virus 2A (Rluc2A-Rep) were electroporated into 8×106 of BHK-21 cells by a Gene Pulser Xcell apparatus (Bio-Rad) at 850 V and 25 μF (23, 24). Then 4×105 and 2×105 transfected cells were seeded in a 12-well plate per well for assaying luciferase signals at 0-24 h post-transfection (p.t.) and at 24-48 h p.t., respectively. At various time points, the cells were washed once with cold PBS, and treated with 250μL of 1×lysis buffer (Promega). The plates containing the lysis buffer were sealed with Parafilm and stored at -80℃. Once samples for all time points had been collected, 20μL of cell lysis was transferred to the 96-well plates, and assayed for luciferase signals in a Turner BioSystems luminometer (Promega).
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Ten microliters of cell lysis, collected at various time points after replicon transfection, were separated on an SDS-PAGE. The proteins in the SDS-PAGE were transferred to a Hybond-C Super nitrocellulose membrane (Amersham). The nitrocellulose membrane was blocked with 5% skim milk in PBS, incubated with primary antibody (anti-actin and anti-WNV NS5) and a secondary antibody (HRP-conjugated goat anti-mouse IgG; Jackson Immuno-Research Laboratories), and developed with chemiluminescence ECL reagents (Amersham).
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Ten micrograms of WNV (strain 3356), YFV (17D vaccine strain), or DENV-1 (Western Pacific strain) replicon (Rluc2A-Rep) were individually electroporated into 8×106 BHK-21 cells. An alphavirus Semliki Forest virus (SFV) replicon was used to express flavivirus structural genes (SFV-CprME-Rep) (23, 24). At 24 h p.t., the cells were electroporated again with 10μg of SFV-CprME-Rep RNAs. At 24 h post the second transfection, the culture fluids were harvested, centrifuged at 415 g for 5 min at 4℃ (to remove cell debris), aliquoted, and stored at -80℃. Both flavivirus Rluc2A-Rep RNA and SFV-CprME-Rep RNA were in vitro transcribed from linearized DNAs, using a T7 and SP6 mMESSAGE mMACHINE kit (Ambion), respectively.
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Two methods were used to quantify VLPs (24). Method one used luciferase assay to compare VLP titers of the same virus-type; method two used indirect immunofluorescence assay (IFA) to estimate the VLP titers of different virus-types. Briefly, for the luciferase assay, approximately 4×104 Vero cells were seeded per well in a 96-well plate. At 24 h post-seeding, the cells were infected with 200μL VLPs, and incubated in 5% CO2 at 37℃. At 24 h p.i., the cells were washed by 200 μL of cold PBS, treated with 20 μL lysis buffer for 20 minutes on a shaker, and then measured for luciferase signals in a Turner BioSystems luminometer (Promega). For the IFA assay, Vero cells in a four Chamber Slide (Nalge Nunc International) were infected with serial dilutions of VLP samples; IFA was performed on the infected cells at 24h p.i.; and IFA-positive cell foci were counted. The VLP titers were estimated by the number of positive IFA-positive foci, and expressed in focus-forming units (FFU)/mL. Immune mouse ascetic fluid of WNV, DENV-1 and YFV (American Type Culture Collection) and goat anti-mouse immunoglobulin G conjugated with Texas Red were used as primary and secondary antibodies, respectively.
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BHK-21 cells were plated in 12-well plates and infected with WNV VLP at 0.5 FFU per cell. At 1 h p.i., cells were washed three times with 2 mL of PBS. At the indicated time points, total cellular RNA was isolated from infected cells using the RNAeasy kit (Qiagen). The RNA was subjected to RT-PCR (Superscript Ⅲ One-step RT-PCR system; Invitrogen) amplification using two primer sets. One primer set spans the luciferase reporter, and the other primer set amplifies the N-terminal region of viral NS5 (Table 1). The RT-PCR products were analyzed using an agarose gel electrophoresis.
Table 1. Primers used for detection of luciferase reporter and NS5
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Heat-inactivated mouse serum was diluted with BA-1, mixed with an equal volume of WNV suspension (200 PFU), and incubated for 1 h at 37℃. The antibody-virus mixtures were incubated in duplicates with Vero cells in 6-well plates. After adsorption of viruses for 1 h at 37℃, 3 mL of a first layer agar containing 0.6% Oxoid agar, basal medium Eagle with 1% FBS, 0.02% DEAE dextran, and 0.13% NaHCO3 was added to the infected cells. Two days later, 3 mL of a second layer agar containing 1% Noble agar, basal medium Eagle with 1% FBS, 0.02% DEAE dextran, 0.13% NaHCO3, and 0.004% neutral red was added over the first layer. The plates were further incubated for 12 h before plaques were counted (23). Neutralization titers are determined as the highest serum dilution that inhibits the formation of at least 90% of the plaques as compared with mock-treated virus control.
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Serum was heat inactivated at 56℃ for 30 minutes. Ten-fold serial dilution of serum samples was mixed with an equal volume containing 100 FFU VLPs of WNV, YFV, or DENV-1. After incubation at 37℃ for 1h, 4×104 Vero cells in a 96-well plate (seeded one day before VLP infection) were infected with the neutralized VLPs. At 22 h p.i., the 96-well plates were subjected to luciferase assay as described above.