Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

Virology 509 (2017) 260–265

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Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

MARK

⁎

Shollie M. Falkenberg , Rohana P. Dassanayake, John D. Neill, Julia F. Ridpath Ruminant Disease and Immunology Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010, United States

A R T I C L E I N F O

A BS T RAC T

Keywords: BRDC BVDV PrimeFlow RNA Assay BL-3

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. Although, BVDV can be identified readily by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely difficult. Detection at the single lymphoid cell level is important due to the immunomodulation that accompanies BVDV infection. A novel PrimeFlow RNA assay using in-situ detection of BVDV was evaluated. The model used to develop this technique included three BL-3 cell lines with different infection statuses, one not infected with BVDV, one infected with BVDV and one dual infected with BVDV and bovine leukosis virus. Using RNA probes specific for the BVDV-2a Npro-Erns coding region, BVDV RNA was detected from both contaminated BL-3 cell lines by flow cytometry and fluorescent microscopy. This is the first report on in-situ detection of BVDV at the single-cell level.

1. Introduction Bovine viral diarrhea viruses (BVDV) belong to the pestivirus genus within the family Flaviviridae. They are enveloped, positive sense single stranded RNA viruses. In the absence of insertion the BVDV genome is approximately 12.3 Kb in length (Simmonds et al., 2012). The genomic RNA includes 5` and 3` untranslated regions (UTR), which flank a long open reading frame encoding a single viral polyprotein (Collett et al., 1991). The order of proteins within the polyprotein is Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5ANS5B (Collett et al., 1991; Elbers et al., 1996). The structural proteins include the nucleocapsid protein (C), and the three envelope glycoproteins Erns, E1 and E2. Previously, it was demonstrated that two glycoproteins E2 and Erns were associated with intracellular membranes and it was proposed that BVDV is released by budding in to the cisternae of the endoplasmic reticulum (ER) (Grummer et al., 2001). In vivo, BVDV infects lymphoid tissue and is characterized in the periphery by a transient lymphopenia that can be observed by day 3 with recovery starting on day 9 and complete recovery in the periphery generally by day 14 post-infection (Falkenberg et al., 2014). RNA in the white blood cell population can be detected by PCR assays, but this method only provides an overview of BVDV infection and is a generalized method of detection. PCR is a general quantification tool, but does not describe the number of infected cells or which cells potentially have

more virus. Reliable and consistent methods for intracellular staining of BVDV infected lymphocytes for evaluation by flow cytometry have been problematic (Qvist et al., 1990, 1991). While the ability to intracellularly stain BVDV poses a variety of problems, there is no method to quantitate BVDV RNA at the cellular level in specific and distinct cell populations. The lack of methods to evaluate infected lymphocytes poses a problem to understanding disease and the nature of the immunomodulation associated with BVDV infections. Recently a novel flow cytometry-based in-situ technique (Prime Flow RNA Assay, ThermoFisher) was developed that allows for amplification of a single RNA transcript as a detection target. Here we adapted this technique for detection of BVDV RNA in individual lymphoid cells. This technique is a reproducible and reliable method to quantify the number of infected cells, quantitate the amount of virus in an individual cell, and differentiate between cells that contain BVDV viral RNA and uninfected cells. The model used to develop and test this technique included three BL-3 cells lines with three different infection statuses, one was not infected with BVDV, one was infected with BVDV and one was dual infected with BVDV and bovine leukosis virus (BLV). While ELISA and real time PCR detected the presence of BVDV in the two infected cell lines, the PrimeFlow RNA Assay demonstrated differences in the prevalence of cells infected in each cell line and differences in viral load.

⁎ Correspondence to: Ruminant Disease and Immunology Research Unit, National Animal Disease Center, Department of Agriculture, Agricultural Research Service, Ames, IA 50010, United States. E-mail address: [email protected] (S.M. Falkenberg).

http://dx.doi.org/10.1016/j.virol.2017.06.032 Received 5 April 2017; Received in revised form 23 June 2017; Accepted 27 June 2017 Available online 05 July 2017 0042-6822/ Published by Elsevier Inc.

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based on the monoclonal antibody N2 that bound to an epitope in the E2 protein of bovine pestiviruses was used (Bauermann et al., 2013; Ridpath et al., 1994).

2. Materials and methods 2.1. Cell lines and reagents

2.4. Cell preparation for BVDV fluorescent protocols

Two bovine B lymphoma cell lines (BL-3 CRL-8037 and BL-3 CRL2306) were obtained from American Type Culture Collection (ATCC). The BL-3 CRL-8037 cell line is contaminated with BVDV, as noted in the ATCC catalog. The BL-3 CRL-2306 cell line in described in the ATCC catalog as having a BLV contaminant. Analysis in our laboratory detected both a BLV and a BVDV contaminant in this cell line. A BVDV free cell line was derived from BL-3 CRL-8037 cells (NADC-BL3-SF) by limiting dilution. All three BL-3 cell lines were maintained in RPMI1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (PAA Laboratories Inc. Ontario, Canada), L-Glutamine (ThermoFisher Scientific, Waltham, MA), antibiotic-antimycotic and incubated at 37 °C in a humid atmosphere of 5% CO2.

Cell suspensions for each respective BL-3 cell line were centrifuged at 400g for 5 min to pellet the cells. The cell pellets were washed with 5 ml of PBS to remove any residual medium and centrifuged again at 400g for 2 min, twice. The cell pellets were resuspended in the appropriate amount of PBS to adjust the cell numbers to be consistent for each cell line (1 × 107 cells/ml). Cells were pelleted and incubated with fixable viability dye eFluor 450 to identify live/dead (LD) cells as described (eBioscience, San Diego, CA) or resuspended in PBS for nonstained control cells. Cell suspensions were washed twice with stain buffer (BD Biosciences, San Jose, CA), aliquoted and used for either BVDV Ab or PrimeFlow RNA assay.

2.2. BVDV RNA extraction and next generation sequencing 2.5. Fluorescent BVDV Ab staining and flow cytometry For RNA extraction, an aliquot of 140 µl of cell culture suspension was submitted for RNA extraction using QIAcube® (Viral RNA kit) according to the manufacture's recommendations (Qiagen, Valencia, CA). BVDV contamination and quantification was confirmed by a commercial reverse transcription quantitative PCR (RT-qPCR) assay (BVDV VetMax Gold) as described previously (Bauermann et al., 2014) as well as using two published RT-PCR tests that target the 5` untranslated region of the viral genome (5` UTR) for identifying the genotype of the contaminate. The primer sets used to detect BVDV contamination were HCV90- 368 and 324–326 with the reactions being performed as previously described (Ridpath et al., 1994; Vilček et al., 1994). The target region of the BVDV genome (Npro-Erns) for PrimeFlow RNA Assay was identified by next generation sequencing (NGS), as well as confirming the BVDV contaminate was the same in both cell lines (CRL-8037 and CRL-2306). For NGS, host cellular DNA and RNA were removed by incubation of samples with a cocktail of DNases and RNases before purification of viral RNA using QIAamp MinElute Virus spin filter kit as per manufacturer's description (Qiagen). BVDV cDNA synthesis, sequencing and genome assembly were performed as described previously (Neill et al., 2014; Victoria et al., 2008).

Cells stained with LD 450 were centrifuged and resuspended in fixation buffer (StableFix) for 10 min. The intracellular BVDV antigen labeling was performed with BD PhosFlow perm buffer III (BD Biosciences) with fluorescein isothiocyanate directly conjugated antiBVDV porcine polyclonal antiserum as per manufacturers’ instructions (VMRD, Pullman, WA). Two-color flow cytometric analyses was performed using the BD LSRII flow cytometer (BD Biosciences). Fixable viability dye eFluor 450 was excited by a violet laser (405 nm) and emission signal was detected using a 450/50 band pass filter. FITC was excited by a blue laser (488 nm) and the emission signal was detected using a 530/30 long-pass filter. BL-3 cells were visualized in forward and side light scatter and electronic gates were placed to contain most of the live cells at single cell levels. At least 50,000 events were collected for data analysis and relative changes in BL-3 cells for BVDV (FITC) were determined using FlowJo software (FlowJo LLC, Ashland, OR). 2.6. PrimeFlow RNA assay and flow cytometry Npro-Erns RNA sequences of newly sequenced BVDV-2a were used to design gene-specific oligonucleotide (RNA) target probes. Bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific probes were designed and used as a control for the assays. In-situ hybridization for BVDV and GAPDH was performed using probes and reagents supplied with PrimeFlow RNA Assay kit as described by the manufacturer (eBiosceince) after incubation of cells with the fixable viability dye eFluor 450 to identify live/dead cells. Although the positive signals were clearly different from negative controls, fluorescence-minus-one controls were included for all the samples to optimize acquisition gates and compensation for each fluorochrome/channel. We selected high sensitivity Alexa Fluor 647 (Type 1, AF647) for BVDV as well as GAPDH detection since it has been recommended for genes with low or unknown expression levels while Alexa Fluor 750 (Type 6, AF750) which shows intermediate-to-low sensitivity was used to compare fluorescent intensity and sensitivity for GAPDH. Flow cytometric analysis was performed using BD LSRII flow cytometer (BD Biosciences). Fixable viability dye eFluor 450 was excited by a violet laser (405 nm) and emission signal was detected using a 450/50 band pass filter. Both Alexa Fluor 647 and 750 were excited using a red laser at 633 nm and emission signals were collected at 660/20 and 780/60 long-pass detection filter sets, respectively. In addition to fluorescenceminus-one controls, compensation beads provided with the kit were also used to set up compensation for each fluorochrome. BL-3 cells were visualized in forward and side light scatter and electronic gates were placed to contain most of the live cells at single cell levels. At least 50,000 events were collected for data analysis and relative changes in

2.3. RT-qPCR BVDV RNA quantification The BVDV contaminate in the CRL-8037 cell line was isolated from the BL-3 cells to use as a reference virus for RT-qPCR. The cell suspension from the CRL-8037 cell line was subjected to a freeze/thaw cycle (−80 °C). Upon thawing of the sample centrifuged to pellet any cell debris and a 500 µl aliquot of the freeze/thaw lysate was used to inoculate a 10 cm2, 60–70% confluent, flask of MDBK cells. After rocking at 37 °C for 1 h, the inoculum was removed from the cells and replaced with 3 ml of cell culture media. Four days later, the cell culture (including media) was frozen at −80 °C. Upon thawing to 25 °C, 500 µl of the resulting lysate was added to a fresh 10 cm2 flask of MDBK cells. Flasks were rocked for 1 h at 37 °C and 3 ml of cell culture media was added. After incubating for 4 days, RNA was extracted from the culture and tested for BVDV as described (Ridpath et al., 2006). The isolate CRL-8037 BVDV was propagated in bovine turbinate cells that had been tested and were found to be free of BVDV and HoBilike viruses (Bauermann et al., 2014). Cells were grown in complete cell culture medium composed of minimal essential media (MEM; SigmaAldrich, St. Louis, MO), supplemented as previously described for the RPMI media. Fetal bovine serum was tested and found to be free of BVDV and HoBi-like viruses and antibodies against BVDV or HoBi-like viruses (Bauermann et al., 2014). Viral titer for the CRL-8037 isolate were determined via dilution on bovine turbinate cells (Bauermann et al., 2012). Endpoints were verified by immunoperoxidase staining 261

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positive cells from each cell line, and the CRL-2306 cells that were BVDV positive had a greater mean fluorescent intensity (MFI; 16,826) than the CRL-8037 BVDV positive cells (2328; Table 1). Probes were also designed to detect expression of bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a house-keeping gene and were used as a positive control for validation of the assay. Two different fluorochromes, AF647 and AF750, were used to evaluate differences that may be observed due to fluorescent intensity and signal of the intracellular probes. All cell lines expressed RNA for GAPDH, but the detection of positive cells was influenced by selection of the fluorochrome (Fig. 2). There was positive expression of GAPDH by all cell lines for detection of the AF647 fluorescent probe ( > 98%; Fig. 2A, B and C), whereas there appeared to be lower expression (80– 98%; Fig. 2D, E and F) when using the AF750 fluorescent probe.

BL-3 cells for BVDV (AF647) and GAPDH (AF750) were determined using FlowJo software (FlowJo LLC). The study was run in duplicate on 2 separate days (2 replicates) and the same trends were observed for the ability to detect BVDV viral RNA in the cell lines. 2.7. Immunofluorescent microscopy To evaluate intracellular BVDV and quantity, cells were subjected to immunofluorescent microscopy, all three BL-3 cell lines which were prepared for PrimeFlow RNA Assay as well as cell labeled using directly conjugated polyclonal serum were used for microscopy. In both preparations, 50 µl of cell suspension were adhered on to slides using Cytospin cytocentrifuge at 10.16g for 3 min and coverslips were mounted using ProLong Gold antifade mount medium with DAPI (ThermoFisher) and visualized using Nikon A1R+ Confocal System microscope (Nikon Instruments, Melville, NY). Images were collected using NIS-Elements Advanced Research software and metadata files were saved as proprietary Nikon ND files. Calibration and binary layers were created for DAPI and Alexa Fluor 647 using the software and then selected frames were saved as TIFF format. Final figures were prepared using Adobe Photoshop Elements 11. At least 100 BL-3 cells were counted to determine the percentage of BVDV positive cells and then to calculate mean fluorescent intensities for BVDV fluorescent signal.

3.3. Detection of BVDV by immunofluorescent microscopy All three BL-3 cell lines which were prepared for PrimeFlow RNA Assay as well as incubated with FITC directly conjugated anti-BVDV polyclonal serum were subjected to immunofluorescent microscopy for evaluation of presence and quantification of BVDV. As with flow cytometry, no fluorescence signal was detected by immunofluorescent microscopy in any of the three cell lines when labeled using a commercially available BVDV polyclonal serum (Fig. 3A). In contrast, when cells were incubated with RNA probes (for PrimeFlow), both the CRL-8037 and CRL-2306 cell line were viral RNA positive and detectable by microscopy (Fig. 3B and C). As expected, NADC-BL3SF cell line was negative for viral RNA (Fig. 3A). The proportion of positive cells within a field of view was quantifiable and similar to the proportion of cells positive by flow cytometry. Of the 100 cells counted by DAPI counterstain for each respective cell line CRL-8037 and CRL2306, 60 and 90 cells were positive for BVDV RNA, respectively. The fluorescent signal associated with the BVDV RNA was restricted to the cytoplasm of positive cells. The CRL-2306 cells that were BVDV positive had a greater MFI of 161.99 compared to the CRL-8037 BVDV positive cells that had an MFI of 103.24 (Table 1). The AF647 GAPDH probe was used for assay validation, and all cells counterstained with DAPI were also positive for GAPDH expression. The detection filter set of the fluorescent microscope used was not set up to detect the AF750 probe.

3. Results 3.1. BL-3 cell lines BVDV contamination verification While ATCC described the CRL-8037 to be contaminated with BVDV and the CRL-2306 to be contaminated with BLV, both cell lines yielded positive result using the panpestivirus primers 324–326 and 90–368 (data not shown). The PCR products were submitted to sequencing and phylogenetic analysis revealed that both sequences belong to the BVDV genotype 2. The NADC-BL3-SF cell line was confirmed to be free of any contaminates by PCR and NGS and it was confirmed that the CRL-8037 cell line had a single contaminant of BVDV but the CRL-2306 had dual contaminants of BVDV and BLV. Full length viral sequences were compared for the BVDV contaminants in the CRL-8037 and CRL-2306 cell lines and it was confirmed that the two cell lines were contaminated with the same BVDV-2a isolate. The endpoint viral titer for the BVDV isolated from the CRL-8037 cell line was used as a reference virus and was determined to be 2.4 × 106 TCID 50/ml. A 10-fold viral dilution curve starting at 2.4 × 106 TCID 50/ ml using the CRL-8037 reference virus was included in the RT-qPCR run. Ct values using the commercial RT-qPCR assay (BVDV VetMax Gold) for the cell lines were 22.2 and 24.2 for the CRL-2306 and CRL-8037 cell lines, respectively. The Ct values corresponded to a viral titer of approximately 2.0 × 104 TCID 50/ml for the CRL-2306 cell line and 2.0 × 103 TCID 50/ml for the CRL-8037 cell line as determined by the dilution curve. Based on the associated Ct values from the RT-qPCR assay, the CRL-2306 cell line has more BVDV than the CRL-8037 cell line.

4. Discussion In this study, we describe a novel technique for a fluorescence based in-situ hybridization assay that can be used with flow cytometry (and fluorescent microscopy) to detect and quantify BVDV RNA in lymphocytes. A limited number of studies for flow cytometry detection of intracellular BVDV in lymphocytes have been published (Qvist et al., 1990, 1991). The previously published reports, highlighted the difficulties encountered in detecting BVDV antigen and described methods that require optimization of the fixation and permeabilization parameters to achieve staining. Each of the reported studies used different procedures (Qvist et al., 1991). While these studies have reported the ability to stain for intracellular BVDV, we were not able to repeat the published methods and for this reason tried other methods to fix and permeabilize the cells for staining. Most likely the inability to stain the cells using the polyclonal sera was impacted by the fixation and permeabilization methods. We were unsuccessful using previously published methods as well as using commercially available fixation and permeabilization products recommended for detection of viral antigens by the manufactures of the products, which highlights the difficultly in using MAbs or polyclonal sera for detection of BVDV. The novel assay described herein is a reliable and repeatable procedure that eliminates a need to optimize the assay for different samples. This PrimeFlow RNA Assay based technique provides the opportunity to stain cells for flow cytometry and microscopy and the capacity to

3.2. Detection of BVDV RNA by PrimeFlow RNA assay for flow cytometry No fluorescence was detected for (BVDV) antigens by flow cytometry for all three BL-3 cell lines using the commercially available BVDV polyclonal serum (data not shown). In contrast, when using the PrimeFlow RNA assay, BVDV viral RNA was detected by a set of probes based on the Npro-Erns coding region (Fig. 1). As expected, no BVDV RNA positive cell population was detected in the NADC-BL3-SF cell line (Fig. 1D). There was a population of cells (~ 60%) within the CRL8037 cell line that were BVDV RNA positive (Fig. 1E). As expected, BVDV RNA positive cell population was also detected in CRL-2306 cell line with approximately 90% of the cells containing viral RNA (Fig. 1F). The mean fluorescent intensity (MFI) was also calculated for the RNA 262

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Fig. 1. Flow cytometric detection of BVDV RNA using RNA PrimeFlow Assay. Representative flow cytometry plots for; (A) NADC-BL3-SF, (B) CRL-8037 and (C) CRL-2306 showing fluorescent minus one (FMO) controls and (D) NADC-BL3-SF, (E) CRL-8037 and (F) CRL-2306 BVDV RNA expression differences between the respective cell lines. The gating strategy was established for the FMO control samples and subsequently applied to the BVDV probe (Type 1) samples. The percentage of positive cells expressing BVDV RNA are reported within the established gate for each cell line.

microscopy. Using this technology we were better able to demonstrate that not only could we detect BVDV, but also show that there were more BVDV infected cells in the CRL-2306 cell line as well as a greater amount of BVDV was detected in the cell, indicated by the greater MFI values, as compared to the CRL-8037 cell line. This technique gave us the ability not to only confirm there was more virus, but specifically contribute the amount of virus to be a combination of more infected cells and more virus in the infected cells. Since NGS sequencing confirmed that both cell lines were infected with the same BVDV strain (2a), it would be hypothesized that the differences in viral load would be due to the dual infection with BLV in the CRL-2306 cell line. The value of being able to detect cells that have more virus provides a utility to better characterize susceptible lymphocyte populations during BVDV infections, as well as better describe viral interactions during coinfections. While the probes designed for this study were specifically designed to detect the BVDV viral contaminant in the BL-3 cell lines, potential future uses of this technology would be probes designed to target BVDV subgenotypes to evaluate if there is a mixed infection. Potential considerations when using the technology, is that not each instrument and fluorochrome can be expected to perform identical to each other. While the absolute values for MFI are not identical between flow cytometry and confocal microscopy, the same general trends were observed regardless of the method used to quantify the amount of virus. The set up for the lasers in each instrument for this study differed, which would impact the MFI values and differed between instruments. Consideration should be given to the selection of the fluorochrome for the RNA target, as this is critical for quantification

Table 1 Mean fluorescent intensity (MFI) values for each BVDV infected cell line using flow cytometry and confocal microscopy. MFI-confocal

CRL−8037 CRL−2306

MFI-flow cytometry

Mean

SD

Mean

SD

103.24 161.99

38.43 69.56

2328.00 16,826.00

316.78 227.69

evaluate viral infection at the individual cell level, rather than relying just on PCR to evaluate the amount of virus in infected cells. For this reason we used multiple cell lines that varied in infection status and BVDV viral load to demonstrate the ability to quantitate viral load at the single cell level. The RT-qPCR results demonstrated there was more BVDV in the dual infected CRL-2306 cell line as measured by a lower Ct value (22.2) when compared to the single infected CRL-8037 that had a greater Ct value (24.2). Since differences in BVDV viral load in the single and dual infected cell lines were detected by RT-qPCR, these cells lines were utilized to verify that the PrimeFlow assay could detect and quantitate the differences in viral load as observed by RT-qPCR. The RT-qPCR provides a broad overview for making comparisons of viral load between the cell lines, but the utility of these assays extends farther to more specifically describe the differences due to the ability to quantify the number of infected cells, quantitate the amount of virus in individual cells, as well as differentiate the between infected and uninfected cells. This was demonstrated both by flow cytometry and 263

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Fig. 2. Flow cytometric detection of GAPDH RNA using RNA PrimeFlow Assay. Representative flow cytometry plots for; NADC-BL3-SF, CRL-8037 and CRL-2306 showing GAPDH expression differences between the different GAPDH probes (Type 1 and Type 6). The gating strategy was established for the fluorescent minus one control samples and subsequently applied to the GAPDH probe samples. The percentage of positive cells expressing GAPDH are reported within the established gate for each cell line.

would be used for quantification should have a fluorochrome that is bright on the spectral scale such as AF647. For the purpose of a housekeeping gene or a control gene for assay verification, lower special fluorochromes provide adequate detection limits. These results highlight the ability to use flow cytometric analysis for BVDV detection and quantification of viral RNA in a lymphoid cells at a

and detection of the target. The most distinct fluorochrome (AF647) was chosen for the BVDV probe since it was unknown what the detection limit would be for the viral RNA for these cell lines. Evaluation of the AF647 versus the AF750 fluorochrome for GAPDH did confirm that probe selection could impact the detection limits. Based on these results, the target which could be in a lesser quantity or 264

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Fig. 3. Immunofluorescent microscopic detection of BVDV RNA using RNA PrimeFlow Assay. Representative confocal microscopy images for; (A) NADC-BL3-SF, (B) CRL-8037 and (C) CRL-2306 showing BVDV RNA expression differences between the respective cell lines for individual cells. All three BL-3 cell lines were incubated with probes and reagents supplied with PrimeFlow RNA Assay kit as described in the materials and methods section. bovine serum lots. J. Vet. Diagn. Investig., (1040638713518208). Bauermann, F.V., Flores, E.F., Ridpath, J.F., 2012. Antigenic relationships between Bovine viral diarrhea virus 1 and 2 and HoBi virus possible impacts on diagnosis and control. J. Vet. Diagn. Investig. 24, 253–261. Bauermann, F.V., Ridpath, J.F., Weiblen, R., Flores, E.F., 2013. HoBi-like viruses an emerging group of pestiviruses. J. Vet. Diagn. Investig. 25, 6–15. Collett, M.S., Wiskerchen, M.A., Welniak, E., Belzer, S.K., 1991. Bovine Viral Diarrhea Virus Genomic Organization, Ruminant Pestivirus Infections. Springer, 19–27. Elbers, K., Tautz, N., Becher, P., Stoll, D., Rümenapf, T., Thiel, H.-J., 1996. Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J. Virol. 70, 4131–4135. Falkenberg, S., Johnson, C., Bauermann, F., McGill, J., Palmer, M., Sacco, R., Ridpath, J., 2014. Changes observed in the thymus and lymph nodes 14 days after exposure to BVDV field strains of enhanced or typical virulence in neonatal calves. Vet. Immunol. Immunopathol. 160, 70–80. Grummer, B., Beer, M., Liebler-Tenorio, E., Greiser-Wilke, I., 2001. Localization of viral proteins in cells infected with bovine viral diarrhoea virus. J. General. Virol. 82, 2597–2605. Neill, J.D., Bayles, D.O., Ridpath, J.F., 2014. Simultaneous rapid sequencing of multiple RNA virus genomes. J. Virol. Methods 201, 68–72. Qvist, P., Aasted, B., Bloch, B., Meyling, A., Rønsholt, L., Houe, H., 1990. Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle. Can. J. Vet. Res. 54, 469. Qvist, P., Houe, H., Aasted, B., Meyling, A., 1991. Comparison of flow cytometry and virus isolation in cell culture for identification of cattle persistently infected with bovine viral diarrhea virus. J. Clin. Microbiol. 29, 660–661. Ridpath, J., Bolin, S., Dubovi, E., 1994. Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66–74. Ridpath, J.F., Neill, J.D., Vilcek, S., Dubovi, E.J., Carman, S., 2006. Multiple outbreaks of severe acute BVDV in North America occurring between 1993 and 1995 linked to the same BVDV2 strain. Vet. Microbiol. 114, 196–204. Simmonds, P., Becher, P., Collett, M., Gould, E., Heinz, F., Meyers, G., Monath, T., Pletnev, A., Rice, C., Stiasny, K., Thiel, H.-.J., Bukh, J., 2012. Genus Pestivirus in Ninth Report of the International Committee on Taxonomy ofViruses (ed. Andrew M.Q. King, Michael J. Adams, Eric B. Carstens, Elliot J. Lefkowitz), 971–1014. Victoria, J.G., Kapoor, A., Dupuis, K., Schnurr, D.P., Delwart, E.L., 2008. Rapid identification of known and new RNA viruses from animal tissues. PLoS Pathog. 4, e1000163. Vilček, Š., Herring, A., Herring, J., Nettleton, P., Lowings, J., Paton, D., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309–323.

single cell level that are known to be infected with BVDV. While these lymphoid cell lines provide a control model to verify the sensitivity of the current assay and the ability to correlate the findings with other methods of viral quantification such as RT-qPCR, further studies are still needed to investigate samples collected from in vivo animal studies. Further evaluation to detect viral RNA in primary samples from BVDV infected animals and characterization of different cell subsets that could be affected over the course of infection would be critical in describing immune responses associated with BVDV. Based on these results it would appear that the PrimeFlow assay designed for detection of BVDV RNA provides the possibility for simultaneous quantification of viral replication at the single cell level and represents a novel approach to evaluate viral load in specific PBMC populations in future studies. Competing financial interests The authors declare no competing financial conflicts of interest. Acknowledgments The authors would like to thank Kathy McMullen, Sam Humphrey and Adrienne Shircliff from the NADC for their excellent technical support. This research was conducted at a USDA research facility and all funding was provided through internal USDA (5030-32000-11700D) research dollars. Mention of trade names, proprietary products, or specified equipment do not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that may be suitable. USDA is an Equal Opportunity Employer. References Bauermann, F.V., Flores, E.F., Falkenberg, S.M., Weiblen, R., Ridpath, J.F., 2014. Lack of evidence for the presence of emerging HoBi-like viruses in North American fetal

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