Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses

Maan, Sushila; Rao, S. Venkat; Maan, Narender S.; Anthony, Simon J.; Attoui, Houssam; Samuel, A. R.; Mertens, Peter P. C.

doi:10.1016/j.jviromet.2007.02.016

Cited by 198 publications

(173 citation statements)

References 17 publications

Supporting

Mentioning

171

Contrasting

Order By: Relevance

“…The current approach for BTV and EHDV phylogenetic analysis is to use whole genome sequencing approaches. 18 This technology will rapidly advance our understanding of the molecular epidemiology of these viruses.…”

Section: Discussionmentioning

confidence: 99%

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

et al. 2009

View full text Add to dashboard Cite

Abstract. Bluetongue virus (BTV) causes disease in domestic and wild ruminants and results in significant economic loss. The closely related Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Therefore, rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the early detection of indigenous and exotic BTV and EHDV is described. This sequence data foundation focused on 2 conserved target genes: one that is highly expressed in infected mammalian cells, and the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for both a virus group-comprehensive and virus group-specific gene amplification diagnostic test. This information has been used as the basis for the development of a rapid multiplex BTV-EHDV real-time RT-PCR that detects all known serotypes of both viruses and distinguishes between BTV and EHDV serogroups. The sensitivity of this rapid, single-tube, real-time RT-PCR assay is sufficient for diagnostic application, without the contamination problems associated with standard gel-based RT-PCR, especially nested RT-PCR tests.

show abstract

Section: Discussionmentioning

confidence: 99%

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

et al. 2009

View full text Add to dashboard Cite

show abstract

“…A self-priming anchor-primer was used to ensure full-length first-strand cDNA synthesis in the absence of free-floating primers, preventing any mispriming and nonspecific amplification (Maan et al, 2007). The anchor-primer consisted of 35 base oligonucleotides that had a spacer9 instead of an original C9 (phosphoramidite) spacer between two complementary halves and a phosphorylated 59 terminus (Hokkaido System Science).…”

Section: Methodsmentioning

confidence: 99%

“…PCR amplification of cDNAs was performed using primer 5-15-1 (59-GAGGGATCCAGTTTAGAATCCTCAGAGGTC-39) containing a BamHI restriction site (underlined) (Maan et al, 2007) and KODPlus-Neo polymerase (Toyobo). Amplification was carried out after denaturation at 94 uC for 2 min, followed by 30 cycles of denaturation at 98 uC for 10 s, annealing at 62 uC for 30 s and extension at 68 uC for 30 s per 1 kb and a final extension step at 68 uC for 3 min.…”

Section: Methodsmentioning

confidence: 99%

Isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (Hypsipetes amaurotis) in Japan

Ogasawara

Kikuchi

Kirisawa

2015

Journal of General Virology

View full text Add to dashboard Cite

Five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified in the genus Orthoreovirus. Their genomes each consist of 10 dsRNA segments. A novel orthoreovirus was isolated from the haemorrhagic intestine of a dead brown-eared bulbul (Hypsipetes amaurotis) in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed nonenveloped capsids of~70 nm diameter, which were characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and sC, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV, but was located on a phylogenetic branch different from that of ARV and NBV. The virus had the closest phylogenetic relationship to two reovirus strains: SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The 10 RNA segments had a 39 pentanucleotide sequence (UCAUC-39) conserved amongst all members of the genus Orthoreovirus, and a unique 59 terminal heptasequence (59-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggested that the novel virus might form a new species with the two strains in the genus Orthoreovirus.

show abstract

“…Amplification of the entire S9 segment was conducted using the full-length amplification of cDNA (FLAC) technique [21,25]. Online services (http://blast.ncbi.nlm.…”

Section: Amplification and Analysis Of The S9 Segmentmentioning

confidence: 99%

Structure and function of S9 segment of grass carp reovirus Anhui strain

Jiang

et al. 2017

VirusDis.

View full text Add to dashboard Cite

A highly virulent grass carp reovirus (GCRV) strain, named GCRV-AH528, was recently purified from a diseased grass carp with hemorrhage disease in Anhui, China. GCRV-AH528 S9 segment was 1320 nucleotides in length and encoded a 418 amino acid VP6 protein. BLAST search showed that the VP6 protein owned a conserved domain belonging to the reoviral r2 family. Phylogenetic analysis of VP6 presented that GCRV-AH528 belonged to GCRV genotype II, which was more closely related to Orthoreovirus than GCRV genotype I and genotype III. Further analysis revealed that GCRV-AH528 S9 and mammalian orthoreovirus S8 might have evolved from a common ancestral precursor and have identical mechanism in virus assembly. The expression level of vp6 gene was detected by quantitative real-time PCR (qRT-PCR). Over time, the expression level of vp6 gradually increased in Ctenopharyngodon idellus kidney cells. However, the level of vp6 expression in blood sharply increased at 4-6 days, and then decreased to a low level after GCRV-AH528 challenge (P \ 0.05). The vp6 gene was detected in all tissues examined, whereas at relatively higher levels in blood, kidney, and liver (P \ 0.05). The yeast two-hybrid (Y2H) system was used to identify VP6 self-interaction, while no interaction was detected in VP6-VP6. This study not only revealed the S9 segment structure and expression pattern but also analyzed the VP6 mechanism by yeast hybridization method. The present study provides valuable informations for further experimental design and investigation of VP6 functions.

show abstract

Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses

Cited by 198 publications

References 17 publications

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

Isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (Hypsipetes amaurotis) in Japan

Structure and function of S9 segment of grass carp reovirus Anhui strain

Contact Info

Product

Resources

About