Concentration of acrylamide in a polyacrylamide gel affects VP4 gene coding assignment of group A equine rotavirus strains with P[12] specificity

<p>Abstract</p> <p>Background</p> <p>It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4.</p> <p>Results</p> <p>To determine which genome segment of three group A equine rotavirus strains (H-2, FI-14 and FI-23) with P[12] specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P[12] strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] specificity did not exhibit this phenomenon when the PAGE running conditions were varied.</p> <p>Conclusions</p> <p>The concentration of acrylamide in a PAGE gel affected VP4 gene coding assignment of equine rotavirus strains bearing P[12] specificity.</p

Whole genome sequencing and phylogenetic analysis of \u3ci\u3eBluetongue virus\u3c/i\u3e serotype 2 strains isolated in the Americas including a novel strain from the western United States

Bluetongue is a potentially fatal arboviral disease of domestic and wild ruminants that is characterized by widespread edema and tissue necrosis. Bluetongue virus (BTV) serotypes 10, 11, 13, and 17 occur throughout much of the United States, whereas serotype 2 (BTV-2) was previously only detected in the southeastern United States. Since 1998, 10 other BTV serotypes have also been isolated from ruminants in the southeastern United States. In 2010, BTV-2 was identified in California for the first time, and preliminary sequence analysis indicated that the virus isolate was closely related to BTV strains circulating in the southeastern United States. In the current study, the whole genome sequence of the California strain of BTV-2 was compared with those of other BTV-2 strains in the Americas. The results of the analysis suggest co-circulation of genetically distinct viruses in the southeastern United States, and further suggest that the 2010 western isolate is closely related to southeastern strains of BTV. Although it remains uncertain as to how this novel virus was translocated to California, the findings of the current study underscore the need for ongoing surveillance of this economically important livestock disease

Identification and Differentiation of the Twenty Six Bluetongue Virus Serotypes by RT–PCR Amplification of the Serotype-Specific Genome Segment 2

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT–PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT–PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm)

Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of \u3ci\u3eBluetongue virus\u3c/i\u3e and \u3ci\u3eEpizootic hemorrhagic disease virus\u3c/i\u3e

Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1–24 and EHDV serotypes 1–7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing

Detection of North American West Nile Virus in Animal Tissue by a Reverse Transcription-Nested Polymerase Chain Reaction Assay

A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues

Field isolates of BTV typed using Seg-2 based serotype-specific RTPCR assays.

*<p>Institute for Animal Health, Pirbright (IAH-P) dsRNA virus reference collection number. More information concerning the origins of these isolates is available at <a href="http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-isolates.htm" target="_blank">http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-isolates.htm</a>.</p

Electrophoretic analysis of cDNA products from Seg-2 of BTV reference strains using ‘type-specific’ primer-pairs for individual serotypes (Panels A–F).

<p>Panel A: PCR amplicons were generated from Seg-2 of BTV-1/RSArrrr/01 using primer-pairs ‘1A1’ −1621 bp, ‘1A2’ −864 bp, and ‘1W1’ −1743 bp (lanes 1, 2, and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-2/RSArrrr/02 using primer-pairs ‘2A1’ −1800 bp, ‘2A2’ −2343 bp and ‘2W2’ −1246 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-3/RSArrrr/03 using primer-pairs ‘3W1’ −648 bp, ‘3W2’ −480 bp and ‘3W3’ −652 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-4/RSArrrr/04 using primer-pairs ‘4W1’ −2045 bp, ‘4W4’ −1071 bp and ‘4W5’ −929 bp (lanes 10, 11 and 12 respectively). <b>Panel B:</b> PCR amplicons were generated using primer-pairs ‘5W1’ −1362 bp, ‘5W2’ −1280 bp and ‘5W3’ −2124 bp from Seg-2 of BTV-5/RSArrrr/05 (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-6/RSArrrr/06 using primer-pairs ‘6W1’ −1724 bp, ‘6W3’ −1303 bp and ‘6W4’ −2051 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-7/RSArrrr/07 using primer-pairs ‘7W1’ −1798 bp, ‘7W2’ −1577 bp and ‘7W3’ −1609 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-8/RSArrrr/08 using primer-pairs ‘8W4’ −363 bp, ‘8W5’ −2216 bp and ‘8W6’ −562 bp (lanes 10, 11 and 12 respectively). <b>Panel C:</b> PCR amplicons were generated using primer-pairs ‘9W1’ −1093 bp, ‘9W2’ −961 bp from Seg-2 of BTV-9/RSArrrr/09 (lanes 1 and 2 respectively), and from Seg-2 of BTV-9/BUL1999/01 using primer-pair ‘9E1’ −1105 bp (lane 3). PCR amplicons were generated from Seg-2 of BTV-10/RSArrrr/10 using primer-pairs ‘10W4’ −964 bp, ‘10W5’ −1094 bp and ‘10W6’ −1109 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-11/RSArrrr/11 using primer-pairs ‘11W4’ −1077 bp, ‘11W5’ −1096 bp and ‘11W6’ −355 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-12/RSArrrr/12 using primer-pairs ‘12W1’ −1613 bp, ‘12W2’ −892 bp and ‘12W3’ −1326 bp (lanes 10, 11 and 12 respectively). <b>Panel D:</b> PCR amplicons were generated from Seg-2 of BTV-13/RSArrrr/13 using primer-pairs ‘13W2’ −1323 bp, ‘13W3’ −1236 bp and ‘13W4’ −1655 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-14/RSArrrr/14 using primer-pairs ‘14W1’ −850 bp, ‘14W2’ −1581 bp and ‘14W3’ −849 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-15/RSArrrr/15 using primer-pairs ‘15W1’ −1823 bp, ‘15W2’ −991 bp and ‘15W3’ −1067 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-16/RSArrrr/16 using primer-pairs ‘16A3’ −851 bp, ‘16E2’ −1288 bp (lanes 10 and 11 respectively) and from Seg-2 of BTV-16/NIG1982/10 using primer-pair ‘16W2’ −726 bp (lane 12). <b>Panel E:</b> PCR amplicons were generated from Seg-2 of BTV-17/RSArrrr/17 using primer-pairs ‘17W1’ −1256 bp, ‘17W4’ −689 bp and ‘17W3’ −1090 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-18/RSArrrr/18 using primer-pairs ‘18W1’ −1021 bp, ‘18W4’ −1556 bp and ‘18W3’ −1381 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-19/RSArrrr/19 using primer-pairs ‘19W1’ −1787 bp, ‘19W2’ −1680 bp and ‘19W3’ −1494 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-20/RSArrrr/20 using primer-pairs ‘20E1’ −1324 bp, ‘20E2’ −1257 bp and ‘20E3’ −827 bp (lanes 10, 11 and 12 respectively).<b>Panel F:</b> PCR amplicons were generated from Seg-2 of BTV-21/RSArrrr/21 using primer-pairs, and ‘21E3’ −1524 bp, ‘21E2’ −1726 bp and ‘21E1’ −1320 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-22/RSArrrr/22 using primer-pairs ‘22W1’ −1074 bp, ‘22W2’ −2034 bp and ‘22W3’ −2211 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-23/RSArrrr/23 using primer-pairs ’23E1’ −1548 bp, ‘23E2’ −1623 bp and ‘23E3’ −1421 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-24/RSArrrr/24 using primer-pairs ‘24W1’ −1776 bp, ‘24W2’ −1557 bp and ‘24W3’ −2021 bp (lanes 10, 11 and 12 respectively).Lane M: 1 Kb marker. +C is a positive control using RNA from BTV-6/RSArrrr/06, with primer-pair BTV-6/2/301F & BTV-6/2/790R −1631 bp – <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032601#pone.0032601-Maan3" target="_blank">[21]</a>. −C is a negative water control. For primer position and sequence see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032601#pone.0032601.s002" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032601#pone.0032601.s003" target="_blank">S2</a>. The use of type specific primers for BTV-26 was recently published by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032601#pone.0032601-Maan2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032601#pone.0032601-Maan4" target="_blank">[22]</a>.</p