First Detection of Heartland Virus Bunyaviridae

First Detection Of Heartland Virus Bunyaviridae-PDF Download

  • Date:22 Nov 2020
  • Views:1
  • Downloads:0
  • Pages:14
  • Size:1.37 MB

Share Pdf : First Detection Of Heartland Virus Bunyaviridae

Download and Preview : First Detection Of Heartland Virus Bunyaviridae


Report CopyRight/DMCA Form For : First Detection Of Heartland Virus Bunyaviridae


Transcription:

MATERIALS AND METHODS,Study area, Ticks were collected at three sites sites 1 2a 2b on two farms owned by case patients who became. ill after infection with HRTV in 2009 3 at three sites on two farms not associated with human disease. and at six sites located on five Missouri Department of Conservation areas MCA Figure 1 Table 1. These locations fell within an area composed of four contiguous counties Andrew Holt Nodaway and. Worth Topographically the study area is characterized by limited local relief with elevations ranging. from their lowest along the Missouri River and the Missouri River flood plain 250 m to their highest. in northern Nodaway County 370 m 6 Steep bluffs are found along the fringes of the Missouri River. floodplain but the broader multicounty area consists of plains and open low hills topped with loess soils. formed by historically wide ranging tallgrass prairies The multicounty area is part of the broader. Western Corn Belt Plains ecoregion Little of the natural tallgrass prairie ecosystem remains today the. area is largely composed of cropland with corn soybeans and other feed grains along with small scale. cattle farming Forested areas are primarily composed of white oak red oak woodland and bur oak. mixed woodland occurring along drainages where the slope of the land is too steep for cultivation 7. Tick collections, In 2012 tick collections occurred over 3 week long time periods April 16 20 June 18 22 and. August 6 10 Ticks were collected primarily by flagging and by use of carbon dioxide CO2 baited tick. traps and less frequently by manual removal from domestic animals Flags were made by securing a. reversible summer infant flannel 68 6 cm by 91 4 cm waterproof pad Kmart Hoffman Estates IL to. bamboo poles or wooden utility handles 8 Tick traps were made from 1 89 L plastic food containers with. holes added to dissipate CO2 Tick traps were baited with 0 5 k of dry ice and placed in the center of an. 0 7 m2 piece of white flannel Jo Ann Fabric and Craft Hudson Ohio anchored by rocks or sticks for. a period of 2 3 hr With the permission of the owners ticks were removed from horses and dogs at. farms owned by the two case patients and on one farm not associated with human disease Ticks were. transferred from flags CO2 baited traps and domestic animals into 16 mL glass Wheaton snap cap vials. Fisher Scientific Pittsburg PA with a solid plaster of Paris charcoal base The base was moistened. with a small amount of water and vials were covered with a piece of latex that was stretched over the. mouth of the vial and held in place by the snap lid with the center removed Ticks were inserted through. a small slit cut in the latex Once the collection was completed or the vial filled the latex was rapidly. replaced with fine mesh to block tick exit and allow ventilation Vials were placed in Whirl Pak bags. Daigger Vernon Hills IL with moist cotton balls to maintain humidity and held in a cooler for. transport to the field laboratory For productive CO2 baited traps with large numbers of ticks the cloth. was rapidly folded and placed into a large labeled zip lock bag for transport to the field laboratory 9. Glass field vials and zip lock bags were chilled and ticks transferred to labeled cryotubes. In August large numbers of larval ticks were present and collected on flags and occasionally on. cloths from CO2 traps Larval ticks when abundant were removed from flags and cloths using a new. method developed by W L Nicholson that used a washable sticky lint roller Sticky Buddy. www stickybuddy com Ticks attached to the roller were washed off with warm water into paper. coffee filters supported by plastic coffee filter holders placed over a plastic container to collect water. Ticks moving upward within the coffee filters were washed downward with a water stream from hand. held squirt bottles The bottom portion of the coffee filter with ticks was twisted closed and rapidly cut. or torn and placed into labeled cryotubes Crytotubes from all sources were held on dry ice until shipped. to Centers for Disease Control and Prevention CDC Fort Collins CO for processing. Tick identification and grinding, Ticks were identified to species sex and stage using dissecting microscopes on refrigerated chill. tables using taxonomic keys 10 16 Ticks were grouped into pools by site collection date collection type. species sex and stage Maximum pool size for engorged ticks was one five for deplete adult ticks 25. for nymphs and 100 for larvae Tick pools were homogenized in chilled 2 or 7 mL glass TenBroeck. grinders Fisher Scientific with alundum bedding material Fisher Scientific as an abrasive for 5 10. sec One mL of chilled bovine albumin 1 BA 1 solution17 was added and the ticks ground to. completion The tick homogenates were poured into 2 mL microtubes Axygen Union City CA and. centrifuged at 4 C and 5 013 RCF for 4 min A 125 L aliquot of the clarified supernatant was. transferred to an identically labeled 1 6 mL microfuge tube for RNA DNA extraction. Primer design and selection, For quantitative real time reverse transcription polymerase chain reaction RT PCR primers and. probes were designed to target the Small S segment of the HRTV genome using the Primer Express. software package PE Applied Biosystems Foster City CA Candidate primers and probes were. selected by referring to nucleic acid sequence data that were previously derived from two human isolates. of HRTV MO 4 and MO 7 3 Selected primer probe sets were then evaluated for relative sensitivity by. application to RNAs that were extracted from 10 fold dilutions of the MO 4 strain of the virus Primer. probe sets that demonstrated the highest relative sensitivity for the detection of HRTV were chosen for. application to RNAs that were extracted from collected ticks Table 2. To screen tick pools for divergent HRTV strains and related viruses and to allow for result. confirmation from more than one amplification platform broadly reactive screening and confirmatory. RT PCR primers were also designed according to an alignment of the nonstructural protein NSs open. reading frames ORFs of the S segments of HRTV3 and the related Chinese SFTSV HB 29 strain NC. 018137 using the Megalign function of the Lasergene software package DNASTAR Madison WI. Primer sets that target regions of maximum homology were then evaluated by application to RNAs that. were extracted from HRTV and SFTSV Upon gel based discrimination two primer sets that. demonstrated the ability to amplify target sized virus specific amplicons from both HRTV and SFTSV. RNAs were selected for application in this study Table 2. RNA extraction and virus detection, Viral RNA was extracted from a 100 L aliquot of supernatant taken from each tick pool using the.
Qiagen QIAmp Virus BioRobot 9604 kit Qiagen Inc Valencia CA on a Qiagen BioRobot Universal. platform according to the manufacturer s protocol Quantitative real time RT PCR was performed by. adding 5 L of each extracted RNA to 50 pmol of each primer and 10 pmol of probe included in HRTV. primer probe set 1 Table 2 in a 50 L total reaction using the Qiagen QuantiTect Probe RT PCR kit. Qiagen according to the manufacturer s instructions Reactions were conducted on Bio Rad s CFX96. Touch real time PCR Detection System Bio Rad Hercules CA using the following cycling. conditions 1 cycle of 50 C for 30 min 1 cycle of 95 C for 10 min and 45 cycles of 95 C for 15 sec and. 60 C for 1 min Positive pools were confirmed by re extracting RNA from the original tick homogenate. and performing the quantitative real time RT PCR assay with two primer probe sets primer probe sets 1. and 4 Table 2 using reaction conditions that were described previously in independent reactions. All tick pools were also assayed with broadly reactive screening primers designed as described. previously Table 2 using the standard RT PCR protocol described below to detect related viruses or. divergent HRTV strains, To generate nucleotide sequence data for phylogenetic analyses traditional RT PCR amplification. was conducted on positive pools through the application of confirmatory RT PCR primers Table 2. Fifty pmol of the forward and reverse primer Table 2 and 5 L of RNA were added to a 50 L total. reaction volume using the Qiagen One Step RT PCR Kit Qiagen according to manufacturer s. recommendations Reactions were conducted using the following cycling conditions 1 cycle of 50 C for. 30 min and 95 C for 15 min followed by 55 cycles of 94 C for 30 sec 50 C for 1 min and 72 C for 2. min Reactions were terminated with a final extension step of 72 C for 10 min Five L of product from. each reaction was then evaluated by electrophoresis in a 2 agarose gel in 40 mM Tris acetate 1 mM. EDTA buffer The DNA bands were visualized by ethidium bromide staining and UV trans. illumination A preliminary positive result was determined by the presence of a target sized DNA band. on the gel DNAs were extracted from 670 bp target sized bands using the Qiaquick Gel Extraction Kit. Qiagen To verify the identity of amplified cDNAs 3 2 pmol of the forward and reverse confirmatory. primers Table 2 were added in independent reactions along with 50 ng of purified DNA and ABI. BigDye Terminator V3 1 ready reaction cycle sequencing mix Applied Biosystems Carlsbad CA in a. total volume of 20 L reaction Sequencing reactions were conducted using recommended cycling. conditions Applied Biosystems Nucleotide sequences were determined by running purified. sequencing reactions on the ABI 3130 genetic analyzer Applied Biosystems Using these methods. multiple nucleotide sequences were generated for each positive tick pool as well as for the isolates. derived from these pools Generated nucleotide sequences were confirmed by the re extraction of RNAs. from tick homogenates followed by re amplification of cDNAs and the generation of additional. nucleotide sequences using methods described previously. Analyses of nucleotide sequence data from ticks and comparison to human isolates. Multiple nucleotide sequences were aligned in both the 5 and 3 directions for each positive tick. pool or isolate using SeqMan software DNASTAR Following editing the resultant consensus. sequences were then reviewed for intact ORFs and subjected to National Center for Biotechnology. Information NCBI basic local alignment search tool BLAST analyses to verify the identity of the. cDNAs at the genus level using the EditSeq function of Lasergene software DNASTAR. http blast ncbi nlm nih gov 18 Alignments were conducted using ClustalW MEGA 5 Phylogenetic. analyses were then conducted on the tick derived partial NSs ORFs along with those of other diverse. phleboviruses using MEGA 5 software Figure 2 19 Evolutionary histories were inferred using both. minimum evolution ME and maximum likelihood ML methods with 2 000 replicates for bootstrap. testing of each grouping 19 23 Trees generated by both methods displayed nearly identical topologies. with comparable bootstrap values for groupings therefore the ML tree is displayed here Figure 2. Plaque assays to detect viable virus, Tick homogenates from RT PCR positive samples were tested for the presence of viable virus using. VeroE6 cell culture plaque assay in six well plates similar to a previously published protocol24 for each. RT PCR positive pool two wells were inoculated each with 100 L of clarified supernatant The. second overlay with neutral red was applied on Day 5 post infection Wells were inspected and plaques. counted on Days 6 8 post infection On Day 8 post infection positive wells were harvested in 1 mL. BA 1 supplemented with 20 Fetal Bovine Serum,Mosquito collections identification and testing. Mosquitoes were collected with CDC light traps baited with dry ice from August 6 to 10 2012. Multiple traps were placed at four sites including both case patient farms sites 1 2a 2b and one site. located on Honey Creek Conservation Area 13b Table 1 Figure 1 Mosquitoes were anesthetized with. dry ice each morning placed in labeled cryotubes and held on dry ice until shipped to CDC for. processing, Mosquitoes were identified on refrigerated chill tables using a dissecting microscope and a standard. reference25 and pooled by species sex and site One copper coated BB and 1 75 mL BA 1 were added. to each pool in a 2 0 mL snap cap tube and homogenized using a Qiagen Mixer Mill MM 300 for 4 min. at 25 cycles sec After centrifugation for 3 min at 5 013 RCF an aliquot of supernatant from each pool. was transferred to a 1 7 mL microcentrifuge tube The RNA was extracted from each aliquot in a 96. well format using the Qiagen BioRobot Universal eluted in 100 L of elution buffer and tested for. HRTV by real time RT PCR and with screening primers by standard RT PCR as described previously. for the tick pools Table 2 In addition mosquito pools were screened by standard RT PCR for. flaviviruses using NS5 gene primers FU2 and cFD3 26 alphaviruses Lanciotti R CDC unpublished. primers and viruses of the genus Orthobunyavirus using the multiplex primers and thermocycler. protocol previously described 27 All standard RT PCR products were subjected to electrophoresis in. 1 2 ethidium bromide stained pre cast agarose gels Invitrogen E Gel Pre cast Agarose. Electrophoresis System and evaluated for the presence of appropriately sized bands. Tick collections and abundance, The 56 428 ticks representing three species were collected at 12 sites on nine properties Figure 1.
Table 1 Amblyomma americanum L and Dermacentor variabilis Say accounted for nearly all ticks. traps and less frequently by manual removal from domestic animals Flags were made by securing a Flags were made by securing a reversible summer infant flannel 68 6 cm by 91 4 cm waterproof pad

Related Books