Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Apr;38(4):1527-35.
doi: 10.1128/JCM.38.4.1527-1535.2000.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses

B Linssen  1 , R M Kinney, P Aguilar, K L Russell, D M Watts, O R Kaaden, M Pfeffer
Affiliations

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses

B Linssen et al. J Clin Microbiol. 2000 Apr.

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.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Schematic drawing of the alphavirus genome organization (adapted from reference 39). The subgenomic 26S RNA coding for the viral structural proteins is enlarged and has been drawn to scale. The primers for the specific detection of each equine encephalitis virus by RT-PCR or nested PCR are shown in boxes. Their orientation is depicted by arrows and by their names (c, complementary to the viral RNA, priming the RT). Each primer combination tested is numbered in consecutive order. The letters following the roman numbers indicate the type of reaction as follows: no letter, RT-PCR; n, nested PCR; and sn, seminested PCR (see also Table 3). The size of each amplicon is given in brackets.
FIG. 2
FIG. 2
Ethidium bromide-stained agarose gels showing the results of EEE-specific amplification. A 10-μl aliquot of each reaction was loaded onto 2% agarose gels. Amplicon sizes are depicted in the margin. (A) Amplification of the EEE-specific 464-bp product resulting from RT-PCR II of 10 different EEE strains (see Table 1) and the closely related alphaviruses Buggy Creek (BCR) and Highlands J (HJ), VEE-IAB strain Trinidad donkey (TRD), and WEE strain Fleming. The negative control contained water instead of RNA in the reaction. (B) The EEE-specific 262-bp amplification products resulting from nested PCR IIn of the template cDNA derived from RT-PCR II (combinations II plus IIn) shown in Fig. 2A. The negative control contained 2 μl of the RT-PCR II negative control reaction. (C) Serial dilutions containing 3 × 105 to 3 RNA molecules of EEE strain NJ/60 used as template for EEE-specific RT-PCR II (left) and subsequently performed nested-PCR IIn (right). The 262-bp amplification product was visible on an ethidium bromide-stained agarose gel down to the dilution containing 30 RNA molecules.
FIG. 3
FIG. 3
Ethidium bromide-stained agarose gels showing the results of WEE-specific amplification. A 10-μl aliquot of each reaction was loaded onto 2% agarose gels. Amplicon sizes are depicted in the margins. (A) Amplification of the WEE-specific 354-bp product resulting from RT-PCR I of 10 different WEE strains (see Table 1) and the closely related alphaviruses VEE-IAB strain Trinidad donkey (TRD), VEE-IIIA strain Mucambo (MUC), VEE-IV strain Pixuna (PIX), EEE strain NJ/60, Aura, Babanki (BBK), Buggy Creek (BCK), Highlands J (HJ), and Fort Morgan (FM). The negative control contained water instead of RNA in the reaction. While all 10 WEE strains showed the amplicon with the expected size, MUC, PIX, and BBK showed a band smaller than 354 bp. (B) The WEE-specific 195-bp amplification products resulting from seminested PCR Isn of the amplicons derived from RT-PCR I shown in Fig. 3A (combinations I and Isn). The negative control contained 2 μl of the RT-PCR I negative control reaction. In this subsequent reaction the 195-bp amplicon was visible only for the WEE strains, rendering the I plus Isn combinations WEE specific.
FIG. 4
FIG. 4
Serial dilutions containing 2 × 109 to 2 RNA molecules of WEE strain Fleming used as a template for WEE-specific RT-PCR I (A) and subsequently performed seminested PCR Isn (B). After RT-PCR, the expected 354-bp amplification product was visible down to the reaction containing 2,000 RNA molecules (A). After the subsequently performed seminested PCR Isn, the 195-bp amplicon was visible on an ethidium bromide-stained agarose gel down to the dilution containing 20 RNA molecules, enhancing the detection limit by a factor of 100 (B).
FIG. 5
FIG. 5
Ethidium bromide-stained agarose gels showing the results of VEE-specific amplification. A 10-μl aliquot of each reaction was loaded onto 2% agarose gels. Amplicon sizes are depicted in the margins. (A) Amplification of the VEE-specific 342-bp product resulting from RT-PCR IV (see Table 3) of 12 VEE subtype varieties. The negative control contained water instead of RNA in the reaction. Only the VEE-IAB to -IE and the VEE-II strains showed the specific amplification product. The remaining VEE subtypes displayed a weak band ca. 100 bp shorter than that of the VEE subtypes above. (B) VEE-specific amplification products of 194 bp resulting from seminested PCR IIIsn of the amplicons derived from RT-PCR IV shown in Fig. 5A. The negative control contained 2 μl of the RT-PCR IV negative control reaction. Besides the VEE subtypes demonstrating a specific amplification during RT-PCR IV, three VEE subtypes (IIIB, IIIC, and IV) showed the specific 194-bp amplicon. (C) Serial dilutions containing 2 × 105 to 2 RNA molecules of VEE-IAB strain Trinidad donkey (TRD) used as a template for VEE-specific RT-PCR IV (left) and subsequently performed seminested PCR IIIsn (right). The 194-bp amplification product was visible on an ethidium bromide-stained agarose gel down to the dilution containing 20 RNA molecules of VEE-IAB strain TRD.
FIG. 6
FIG. 6
Ethidium bromide-stained agarose gels showing the results of VEE-specific amplification as applied to the VEE strains and serum samples listed in Table 2. A 10-μl aliquot of each reaction was loaded onto the 2% agarose gels. Amplicon sizes are depicted in the margin. (A) Amplification of the VEE-specific 342-bp product resulting from RT-PCR IV (see Table 3) was successful with RNA of all VEE isolates from Peru and Mexico and with RNA of serum sample IQT 8131. The negative control contained RNA of a Mayaro virus-positive human serum sample in the reaction. (B) In addition to the samples found to be positive by RT-PCR alone, all samples showed the VEE-specific 194-bp amplification products resulting from seminested PCR IIIsn of the amplicons derived from RT-PCR IV shown in Fig. 6A. The negative control contained 2 μl of the RT-PCR IV-negative control reaction.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Armstrong P, Borovsky D, Shope R E, Morris C D, Mitchell C J, Karabatsos N, Komar N, Spielman A. Sensitive and specific colorimetric dot assay to detect eastern equine encephalomyelitis viral RNA in mosquitos (Diptera: Culicidae) after polymerase chain reaction amplification. J Med Entomol. 1995;32:42–52. - PubMed
    1. Brightwell G, Brown J M, Coates D M. Genetic targets for the detection and identification of Venezuelan equine encephalitis viruses. Arch Virol. 1998;143:731–742. - PubMed
    1. Calisher C H, Karabatsos N, Lazuick J S, Monath T P, Wolff K L. Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests. Am J Trop Med Hyg. 1988;38:447–452. - PubMed
    1. Calisher C H, Kinney R M, de Souza Lopes O, Trent D W, Monath T P, France D B. Identification of a new Venezuelan equine encephalitis virus from Brazil. Am J Trop Med Hyg. 1982;31:1260–1272. - PubMed
    1. Calisher C H, Monath T P, Sabattini M S, Cropp C B, Kerschner J, Hunt A R, Lazuick J S. Arbovirus investigations in Argentinia, 1977–1980. III. Identification and characterization of viruses isolated, including new subtypes of western and Venezuelan equine encephalitis viruses and four new bunyaviruses (Las Malayos, Resistencia, Barranqueras, and Antequera) Am J Trop Med Hyg. 1985;34:956–965. - PubMed

Publication types

MeSH terms

LinkOut - more resources