Genetic targets for the detection and identification of Venezuelan equine encephalitis viruses

Brightwell, Gale; Brown, Joseph; Coates, D. M.

doi:10.1007/s007050050326

Cited by 11 publications

(14 citation statements)

References 19 publications

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“…This is in contrast to the EEE-specific assay described by Armstrong and coworkers, which failed to detect the RNA of EEE strains from Panama and Brazil (16). Because there was no heterologous amplification in the subsequent nested PCRs, we did not investigate the origin of the nonspecific Aura virus-derived amplicon in the EEE-specific RT-PCR I or the reaction of the MUC, PIX, and BBK strains with WEE RT-PCR I. Amplicons that differ in size from the specific PCR product have been observed with various bacterial DNAs or mosquito-borne flaviviral RNAs in a recently described panel of VEE-specific RT-PCR assays (2). Such circumstances require additional tests to confirm the amplicon's origin.…”

Section: Discussionmentioning

confidence: 99%

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Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses

et al. 2000

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Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR–seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR–seminested PCR.

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Section: Discussionmentioning

confidence: 99%

“…RT-PCR is a fast, sensitive, and specific alternative for the diagnosis of infections caused by RNA viruses (26). This technique has been described for the detection of EEE (1,40), WEE (41), and VEE (2). In those studies, the specificity of the RT-PCRs was not thoroughly tested.…”

Section: Specific and Sensitive Reverse Transcription-pcr (Rt-pcr) Asmentioning

confidence: 99%

Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses

et al. 2000

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“…While the lack of nucleotide sequence conservation renders the C-terminal nsP3 coding region unreliable for RT-PCR diagnosis of VEE virus infection using nondegenerate primers (Brightwell et al, 1998), we had no difficulty amplifying the C-terminal regions of all strains using a single degenerate primer pair hybridizing to neighboring conserved regions. Because nucleotide sequences of the C-terminal nsP3 region distinguish between even closely-related strains, the diagnostic utility of this region is obvious.…”

Section: Discussionmentioning

confidence: 99%

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex

et al. 1999

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The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.

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“…Antigenbased methods are unavailable, and although serology can confirm the diagnosis, this requires paired acute and convalescent samples [2]. Current molecular tests lack optimal performance metrics characteristics necessary for routine diagnostic testing [10][11][12][13][14][15][16]. Molecular assay designs have been complicated by the genetic variability of the VEE complex viruses, which comprise of at least 14 different viral subtypes [1].…”

Section: Introductionmentioning

confidence: 99%

Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses genome detection in human and mosquito populations

Carrera

Araúz²,

Rojas

et al. 2022

Preprint

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Background. Venezuelan equine encephalitis (VEE) complex, eastern equine encephalitis virus (EEEV), and Maradiaga virus (MADV), are associated with severe neurological disease in human and equine hosts. We aimed to overcome the lack of available molecular diagnostics for these pathogens by designing and clinically evaluating real-time reverse transcription PCRs (rRT-PCRs) for 1) VEE complex and 2) MADV/EEEV. Methodology/Principal Findings. Primers and probes were designed from alignments of all publicly available complete genome sequences for VEE complex, EEEV and MADV. Linear range for the VEE complex and MADV/EEEV rRT-PCRs extended from 2.0 to 8.0 log10 copies/uL with exclusive detection of the respective viruses. Fifteen serum samples from febrile patients collected from 2015 and 2017 outbreaks in Panama were tested to evaluate the new assays. Ten samples (66.7%) samples were positive for VEEV, and in one sample, rRT-PCR detected both VEEV and MADV. Early acute samples (≤ 3 days since onset) with coincidental anti-VEEV IgM and IgG antibodies were also found positive for VEEV viral RNA. Of 150 mosquito pools, 3 tested positive for VEEV by rRT-PCR. Two of these also yielded VEEV isolates. Conclusions. The VEE complex and MADV/EEEV rRT-PCRs provide accurate detection while yielding significant benefits over available molecular methods. Data from the clinical evaluation provide further evidence for VEE complex and MADV co-infections and suggest that re-infection with VEE complex viruses is possible.

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Genetic targets for the detection and identification of Venezuelan equine encephalitis viruses

Cited by 11 publications

References 19 publications

Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses

Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex

Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses genome detection in human and mosquito populations

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