Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA

In 2018, West Nile virus (WNV) broke out for the first time in Germany, with continuation of the epidemic in 2019, involving birds, horses and humans. To identify vectors and characterize the virus, mosquitoes were collected in both years in zoological gardens and on a horse meadow immediately following the diagnosis of disease cases in birds and horses. Mosquitoes were identified and screened for WNV by qRT-PCR, with virus-positive samples being sequenced for the viral envelope protein gene. While no positive mosquitoes were found in 2018, seven mosquito pools tested positive for WNV in 2019 in the Tierpark (Wildlife Park) Berlin. The pools consisted of Cx. pipiens biotype pipiens (n = 5), and a mixture of Cx. p. biotype pipiens and Cx. p. biotype molestus (n = 2), or hybrids of these, and were collected between 13 August and 24 September 2019. The virus strain turned out to be nearly identical to two WNV strains isolated from birds diseased in 2018 in eastern Germany. The findings represent the first demonstration of WNV in mosquitoes in Germany and include the possibility of local overwintering of the virus.

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Establishment and evaluation of real-time PCR for West Nile virus detection

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236209002599 ◽

2009 ◽

Vol 6 (1) ◽

pp. 55-59 ◽

Cited By ~ 1

Author(s):

Shi Li-Jun ◽

Lu Mao-Min ◽

Li Gang ◽

Li Cheng-Yao ◽

Zhang Jin-Gang

Keyword(s):

West Nile Virus ◽

Real Time ◽

Virus Detection ◽

Rt Pcr ◽

Pcr Assay ◽

West Nile ◽

Capsid Protein Gene ◽

Specific Test ◽

Intercalating Dye ◽

Polymerase Chain

AbstractA rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

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Detection of West Nile virus lineages 1 and 2 by real-time PCR

Journal of Virological Methods ◽

10.1016/j.jviromet.2007.05.021 ◽

2007 ◽

Vol 146 (1-2) ◽

pp. 355-358 ◽

Cited By ~ 77

Author(s):

Sonja Linke ◽

Heinz Ellerbrok ◽

Matthias Niedrig ◽

Andreas Nitsche ◽

Georg Pauli

Keyword(s):

West Nile Virus ◽

Real Time ◽

Real Time Pcr ◽

West Nile

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

Journal of General Virology ◽

10.1099/vir.0.82552-0 ◽

2007 ◽

Vol 88 (4) ◽

pp. 1163-1168 ◽

Cited By ~ 46

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.

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