Genus-Specific Detection of Alphaviruses by a Semi-Nested Reverse Transcription-Polymerase Chain Reaction

Pfeffer, Martin; Proebster, Barbara; Kinney, Richard M.; Kaaden, Oskar‐Rüger

doi:10.4269/ajtmh.1997.57.709

Cited by 114 publications

(94 citation statements)

References 17 publications

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“…As both alphaviruses and flaviviruses were reported in the region of Kerala, PCR was initially performed to characterize these two genera of arboviruses. The multiplex primers used were forward M2W and reverse cM3W primers (Pfeffer et al, 1997), and forward FG1 and reverse FG2 primers (Fulop et al, 1993), which amplify the nonstructural protein 1 (NSP1) gene of Alphaviruses and the nonstructural protein 5 (NS5) gene of Flaviviruses, respectively. As these primers do not amplify the gene for CHIKV, we also included a CHIKV non-structural protein 1 (NSP1) primer set (Hasebe et …”

Section: Methodsmentioning

confidence: 99%

Genetic characterization of dengue viruses prevalent in Kerala State, India

Kumar

Jayakumar

George

et al. 2013

Journal of Medical Microbiology

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Section: Methodsmentioning

confidence: 99%

Genetic characterization of dengue viruses prevalent in Kerala State, India

Kumar

Jayakumar

George

et al. 2013

Journal of Medical Microbiology

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“…Viral RNA was extracted by using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA), and cDNA was synthesized by using Ready-To-Go You-Prime First Strand Beads (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's procedure. Samples were tested by polymerase chain reaction (PCR) with flavivirus-specific primers, 19 alphavirus-specific primers 20 and primers to detect the SINV nonstructural protein 2 (NS2) gene, the JEV envelope (E) gene, the 12th segment of Banna virus (BAV), 21 the VP7 gene of Yunnan orbivirus (YUOV), 22 and the densovirus (DNV) partial NS1 gene 15 ( Table 1 ). The reverse transcription-PCR conditions were 1 hour at 37°C, 5 min at 94°C, and 35 cycles of amplification (30 seconds at 94°C, 30 seconds at 55°C, and 2 minutes at 72°C), and final extension at 72°C for 10 minutes.…”

Section: Mosquitomentioning

confidence: 99%

“…The results show that of the 184 serum samples obtained from hospitalized patients, 26.6% (49 of 184) were positive for IgM against JEV ( Table 4 ). Of these patients, 19.6% (20 of 102) had fever and 35.3% (29 of /82) had encephalitis. The results suggest that JEV infection persists in the region.…”

Section: Banna Virusmentioning

confidence: 99%

Distribution of Mosquitoes and Mosquito-Borne Arboviruses in Yunnan Province near the China–Myanmar–Laos Border

Wang¹,

Zhang²,

Sun³

et al. 2011

The American Society of Tropical Medicine and Hygiene

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Abstract. Economic development and increased tourism in the southern region of Yunnan Province in China, adjacent to several countries in Southeast Asia, has increased the likelihood of import and export of vectors and vector-borne diseases. We report the results of surveillance of mosquitoes and mosquito-borne arboviruses along the border of ChinaMyanmar-Laos in 2005 and 2006, and information associating several arboviruses with infections and possibly disease in local human populations. Seventeen mosquito species representing four genera were obtained, and 14 strains of mosquito-borne viruses representing six viruses in five genera were isolated from Culex tritaeniorhynchus . In addition, IgM against Japanese encephalitis virus, Sindbis virus, Yunnan orbivirus and novel Banna virus was detected in acute-phase serum samples obtained from hospitalized patients with fever and encephalitis near the areas where the viruses were isolated. This investigation suggests that Japanese encephalitis virus, Sindbis virus, and lesser-known arboviruses circulate and may be infecting humans in the China-Myanmar-Laos border region. immune ascites against alphaviruses, specific antisera to SINV, Getah virus, Mayaro virus, chikungunya virus, and Simliki Forest virus, and antisera against the flaviviruses JEV, dengue virus, and West Nile virus. Antisera were diluted 1:50 in PBS before use. After subsequent rinses in PBS and sterile water, the slides were probed with the secondary rabbit anti-mouse monoclonal fluorescein isothiocyanate conjugate (Sigma, St. Louis, MO) at 37°C for 30 minutes before visualization with a fluorescent microscopy. Positive and negative controls of immune ascites against alphaviruses were SINV (YN87448) and JEV (P3), respectively. Positive and negative controls of immune ascites against flavivirus antisera were JEV (P3) and SINV (YN87448), respectively.Polyacrylamide gel electrophoresis, RNA extraction, cDNA synthesis, reverse transcription-polymerase chain reaction, and sequence analysis. RNA extraction and polyacrylamide gel electrophoresis were performed as described. 18 Briefly, doublestranded RNA was extracted from approximately 400 μL of cell suspension with phenol/chloroform. Each RNA sample was mixed with sample buffer and subjected to electrophoresis at room temperature on a standard discontinuous 7%, 10%, or 15% acrylamide (acrylamide/bisacrylamide 29:1; Bio-Rad Laboratories, Hercules, CA) slab gel (18 × 16 × 0.075 cm) (Hoefer Pharmacia Biotech Inc., San Francisco, CA) with a 3.5% acrylamide stacking gel in Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) (Bio-Rad Laboratories). After electrophoresis, virus double-stranded RNAs were visualized by staining with silver nitrate. Viral RNA was extracted by using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA), and cDNA was synthesized by using Ready-To-Go You-Prime First Strand Beads (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's procedure. Samples were tested by polymerase chain reaction (PCR) with flavivi...

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“…The prepared rnA was used as the template for cDnA synthesis using the Maxime™ rt PreMix (Intron Biotechnology, rok) according to the manufacturer΄s protocols. Pcr reactions were carried out by previously described methods (14). After cloning Pcr products into pluG® Multi tA-cloning vector (Intron Biotechnology, rok), the sequences of purified clones were analyzed by Macrogen (rok).…”

mentioning

confidence: 99%

Characterization of recent Getah virus isolates from South Korea

Hj¹,

Hc²,

Ta³

et al. 2012

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Genus-Specific Detection of Alphaviruses by a Semi-Nested Reverse Transcription-Polymerase Chain Reaction

Cited by 114 publications

References 17 publications

Genetic characterization of dengue viruses prevalent in Kerala State, India

Genetic characterization of dengue viruses prevalent in Kerala State, India

Distribution of Mosquitoes and Mosquito-Borne Arboviruses in Yunnan Province near the China–Myanmar–Laos Border

Characterization of recent Getah virus isolates from South Korea

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