A simple and rapid approach to prepare Sindbis and West Nile viral RNA controls for differentiation between positive samples and laboratory contamination

2020 ◽  
Vol 278 ◽  
pp. 113822
Author(s):  
Micah Dimaculangan ◽  
Siewert C. Wiid ◽  
Phillip A. Bester ◽  
Tumelo R. Sekee ◽  
Felicity J. Burt
Keyword(s):  
Viral Rna ◽  
West Nile ◽  
Laboratory Contamination

scholarly journals West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis

2007 ◽  
Vol 88 (4) ◽  
pp. 1163-1168 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Mark T. Kenney ◽  
Edwin G. Westaway
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Virus Strain ◽  
Viral Rna ◽  
West Nile ◽  
Replication Complex ◽  
Virus Rna ◽  
West Nile Virus Strain ◽  
First Time ◽  
Cytoplasmic Location

Using West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.

scholarly journals Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 11989-12000 ◽  
Author(s):  
W. Li ◽  
Y. Li ◽  
N. Kedersha ◽  
P. Anderson ◽  
M. Emara ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
Peptide Sequencing ◽  
Viral Rna ◽  
Cell Protein ◽  
Affinity Column ◽  
Rna Recognition Motif ◽  
The West ◽  
West Nile ◽  
Stem Loop

ABSTRACT It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (Kd ) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10−8, while that for TIA-1 RRM2 was 1.12 × 10−7. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

scholarly journals THE INFORMATIVE VALUE OF IMMUNOLOGICAL AND GENETIC TESTS IN THE DIAGNOSIS WEST NILE FEVER

2020 ◽  
Vol 76 (4) ◽  
pp. 42-45
Author(s):  
Т.V. Zamarina ◽  
◽  
N.P. Khrapova ◽  
I.A. Barkova ◽  
E.V. Pimenova ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Rna ◽  
Unknown Etiology ◽  
West Nile Fever ◽  
West Nile ◽  
Respiratory Viral Infection ◽  
Blood Samples ◽  
Monitoring Centre ◽  
Laboratory Confirmation ◽  
Acute Respiratory Viral Infection

We used ELISA and PCR for laboratory verification of West Nile fever (MUC 4.2.3009-12). We analyzed serum and whole blood samples which had been sent to West Nile fever reference monitoring centre in 2018–2019. A total of 270 blood samples obtained from patients with presumed viral encephalitis of unknown etiology, acute respiratory viral infection, acute respiratory viral infection, meningitis, acute gastroenteritis were analyzed. Antibodies against WNV were detected in 193 (71,4 %) blood samples, while 146 samples were found to be capable of developing an immune response with viral RNA not being detected in them. Both WNV antibodies and viral RNA were detected in 47 (17,4 %) samples tested. WNV RNA was detected in only 14 (5,1 %) cases. Thus, laboratory confirmation of WNF was obtained in 76,6 % of cases (207 out of 270).

scholarly journals Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Noelle Fynmore ◽  
Renke Lühken ◽  
Heike Maisch ◽  
Tina Risch ◽  
Sabine Merz ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rapid Assessment ◽  
Gravid Female ◽  
Virus Detection ◽  
Turnaround Time ◽  
Viral Rna ◽  
Mass Trapping ◽  
Vector Surveillance ◽  
West Nile ◽  
Fta Cards

Abstract Background For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. Methods In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. Results A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. Conclusion The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory. Graphical Abstract

scholarly journals West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Justin T. O’Neal ◽  
Amit A. Upadhyay ◽  
Amber Wolabaugh ◽  
Nirav B. Patel ◽  
Steven E. Bosinger ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Single Cell ◽  
Type I Interferon ◽  
Single Cells ◽  
Antiviral Response ◽  
Viral Rna ◽  
Type I ◽  
Cell Level ◽  
West Nile ◽  
Viral Abundance

ABSTRACTWest Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCEWest Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells orin vivomodels to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

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