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

Abstract

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.

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses

“…Molecular detection by RT-PCR is an ideal method for detection of these viruses in hosts because it is rapid, sensitive, specific, reproducible, and amenable to automation. RT-PCR has been used for the detection of arboviruses at the group level (Bronzoni et al, 2004;Pfeffer et al, 1997;Sanchez-Seco et al, 2001;Scaramozzino et al, 2001) and for specific detection of individual arboviruses (Lambert et al, 2003;Lanciotti and Kerst, 2001;Lee et al, 2002;Linssen et al, 2000;O'Guinn et al, 2004), as well as for a sequential detection at both levels (Bronzoni et al, 2005).…”

Sensitive and specific detection of strains of Japanese encephalitis virus using a one‐step TaqMan RT‐PCR technique

“…The average time required for the laboratory confirmation of JEV is approximately 7 days in Taiwan. A rapid and accurate The virus titer for most of these viruses has not been well quantified before JEV NS3 TaqMan assay; however, these viral RNAs have been checked by their virus specific primer pairs as described [Dennis et al, 1992;Linssen et al, 2000]. All of these viruses have clear and distinct bands of RT-PCR product with expected sizes.…”

Magnetic Nanotrap Particles Preserve the Stability of Venezuelan Equine Encephalitis Virus in Blood for Laboratory Detection

“…Reactions based on amplification of the nucleic acid is often used in fatal cases to confirm pathogen presence (5,19,31). RT-qPCR is a very reliable method and highly sensitive for the detection of minimal amounts of viral nucleic acids in the patient samples (11). However, this method is based on the detection of relatively short fragments of the viral genome and analysis of a longer stretch of viral RNA (vRNA) would provide a more stringent measure of RNA stability.…”

“…Viral stability is an essential factor for diagnosis of VEEV to confirm the presence of virions or viral RNA in a clinical sample. The current diagnostic approaches to confirm VEEV infection in humans or horses rely on direct detection of viral nucleic acids in serum or spinal fluid samples during the acute-phase of infection using reverse-transcription PCR (RT-PCR) (11) and ELISA for VEEV-specific IgM (12). However, despite high sensitivity of the above-mentioned methods, false negative results can be obtained if samples have been collected during the initial asymptomatic phase of infection where the viral load is low (13)(14)(15).…”