Propagation, Quantification, Detection, and Storage of West Nile Virus

Brien, James D.; Lazear, Helen M.; Diamond, Michael S.

doi:10.1002/9780471729259.mc15d03s31

Cited by 117 publications

(143 citation statements)

References 31 publications

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“…The viral supernatants were harvested at 24 h and 48 h posttransfection. For production of prM-E virus particles, the 293T-Lenti-prM-E bulk cell line was cultured in the absence of blasticidin, and supernatants were harvested at 72 h and 96 h. VLPs were concentrated per the protocol of Brien et al (49). Harvested supernatants (25 to 30 ml) were transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer (10 mM Tris-Cl, 150 mM NaCl, 1mM EDTA).…”

Section: Discussionmentioning

confidence: 99%

“…Hence, for generation of C-prM-E VLPs, cells were cotransfected with the NS2B-3 expression plasmid (52). Using these two different strategies, we generated both prM-E and C-prM-E VLPs that were purified from culture supernatants by ultracentrifugation through a glycerol cushion (49). The purified VLP pellet was analyzed for E protein expression via Western blotting and for VLP morphology by electron microscopy.…”

Section: Figmentioning

confidence: 99%

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Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Garg

Sedano

Plata

et al. 2017

J Virol

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Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. We used a virus-like particle (VLP)-based approach to develop a vaccine and a microneutralization assay for ZIKV. A synthetic capsid-premembrane-envelope (CprM-E) gene construct of ZIKV was used to generate reporter virus particles (RVPs) that package a green fluorescent protein (GFP) reporter-expressing West Nile virus (WNV) replicon. The assay was adapted to a 96-well format, similar to the plaque reduction neutralization test (PRNT), and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While the ZIKV prM-E construct alone was sufficient for generating VLPs, efficient VLP production from the C-prM-E construct could be achieved in the presence of the WNV NS2B-3 protease, which cleaves C from prM, allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than those with the DNA vaccines, with C-prM-E VLPs giving slightly higher titers than those with prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective, and economical VLP-based vaccine against ZIKV.IMPORTANCE To address the growing Zika virus epidemic, we undertook this study with two objectives: first, to develop a safe, effective, and economical vaccine for ZIKV, and second, to develop a rapid and versatile assay to detect the anti-ZIKV immune response. We generated a cell line stably expressing ZIKV prM-E that produces large amounts of VLPs in the supernatant and a ZIKV C-prM-E cell line that produces reporter virus particles upon transfection with a GFP replicon plasmid. The prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid was included. VLP-based vaccines showed significantly better neutralizing antibody responses than those with their DNA counterparts. The RVP-based microneutralization assay worked similarly to the PRNT assay, with a rapid GFP readout in a 96-well format. Our VLP-based platform provides a source for a ZIKV vaccine and diagnosis that can rapidly be adapted to current outbreaks.KEYWORDS C-prM-E, diagnostics, microneutralization, neutralization, PRNT, reporter, reporter virus particles, vaccine, Zika, prM-E S ince the identification of Zika virus (ZIKV) from a rhesus monkey in Uganda in 1947 (1, 2) until 2010, the virus predominantly circulated between Aedes mosquitoes and nonhuman primates. Periodic episodes were inde...

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Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Garg

Sedano

Plata

et al. 2017

J Virol

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“…WNV (strain 3000.0259, isolated in New York in 2000) (64) was used in all experiments except those depicted in Supplemental Figure 5, in which the WNV-Madagascar (strain DakAnMg798, isolated in 1978) (65) also was used. Viral titers in all studies were assessed via plaque assay in BHK21-15 cells, as previously described (66).…”

Section: Methodsmentioning

confidence: 99%

“…Primers used for all analyses are reported in Supplemental Table 4. qRT-PCR analysis of WNV copy numbers in serum and cell culture supernatants was performed with TaqMan reagents (Thermo Fisher Scientific) as previously described (27,66).…”

Section: Methodsmentioning

confidence: 99%

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection

Daniels¹,

Jujjavarapu²,

Durrant³

et al. 2017

Journal of Clinical Investigation

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110

112

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“…BHK21 (C-13; American Type Culture Collection [ATCC], Manassas, VA) and Vero E6 (ATCC) cell lines, which are permissive to WNV infection (12), were used to isolate WNV from the urine samples. Cells were grown in three different cell culture supports, i.e., shell vials, 6-well plates, and T25 tissue culture flasks.…”

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confidence: 99%

Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

et al. 2014

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cThis study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified. West Nile virus (WNV), a mosquito-borne flavivirus, has become a public health emergence in recent years, since it has been responsible for large outbreaks of neuroinvasive disease and fever in humans in many European countries and in America (1). In humans, WNV infection is asymptomatic in approximately 80% of cases, while it presents as West Nile fever (WN-F) in 20% of cases and as neuroinvasive disease (WN-ND) in less than 1% of cases (2).Laboratory diagnosis of acute WNV infection is based on the detection of WNV RNA in blood and cerebrospinal fluid (CSF), viral isolation in cell culture, and the demonstration of WNVspecific antibodies. This laboratory diagnosis is challenging because the WNV load in blood is generally very low or undetectable at the time of symptom onset and because WNV antibodies crossreact with other flaviviruses, thus requiring confirmation by neutralization assays. A relevant improvement to the diagnosis of WNV infection was recently provided by WNV RNA testing in urine, since viral RNA is detectable in urine at a higher load and for a longer time than in blood or CSF (3-11). In addition, the isolation of infectious WNV in cell cultures from urine samples has been reported in some patients with acute infection (8-10). However, the protocols for WNV isolation from urine have not been standardized, and the infectivity of WNV excreted in urine has not been investigated in detail. Thus, the aim of this study was to investigate the infectivity of WNV excreted in urine from patients with acute infection. To this aim, methods for the isolation of WNV in cell cultures from urine samples were set up.The urine samples investigated in this study were collected from 17 patients from northern Italy with acute WNV infection in 2013. The cases included 7 patients with WN-ND, 6 patients with WN-F, and 4 asymptomatic blood donors in whom WNV RNA was detected in urine by real-time reverse transcription (RT)-PCR, according to previously reported methods (8). Thirteen patients were infected with WNV lineage 2 (Italy/2013 strain), and four were infected with WNV lineage 1 (Livenza strain). Genome sequence information on these strains was previously reported (9). Urine samples were stored at 2 to 8°C for up to 72 h and at Ϫ80°C thereafter. This study was approved by the institutional review board and ethics committee of Padua University Hospital.All of the procedures for WNV isolation were performed within certified biosafety cabinets under biosafety level 3 (BSL3) containment. BHK21 (C-13; American Type Culture Collection [ATCC], Manassas, VA) and Vero E6 (ATCC) cell lines, which are permissive to WNV infection (12), were used to isolate WNV from the urine samples. Cells were grown in three different cell cultur...

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Propagation, Quantification, Detection, and Storage of West Nile Virus

Cited by 117 publications

References 31 publications

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection

Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

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