Viruses and cells
The isolates SD16-38 and SD16-49 were obtained in 2016 at Animal Disease Research and Diagnostic Laboratory at South Dakota State University (ADRDL-SDSU) using bovine turbinate (BT) cells (ATCC; CRL-1390). BoHV-4 SD16-49 was isolated from a cow with a reproductive disorder; isolate SD16-38 was recovered from lung tissue from a calf with respiratory disease. The isolate SD16-49 was further amplified and titrated using Madin-Darby Bovine kidney cells (MDBK, ATCC, CCL-22). Whereas bovine turbinate cells (BT) were used for amplification and titrations of isolate SD16-38. Cells were tested free of non-cytopathic bovine viral diarrhea virus (BVDV). Cells were cultured at 37 °C with 5% CO2 in minimum essential medium (MEM) (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS; VWR, Radnor, PA), penicillin (100 IU/ml), streptomycin (100 μg/ml), and gentamicin (50 μg/ml) (Corning, Corning, NY).
Whole genome sequence and characterization
Supernatants of BoHV-4-inoculated cell monolayers (SD16-38 and SD16-49) were subjected to high throughput sequencing using the Illumina MiSeq sequencing platform. DNA libraries were prepared using the Nextera XT DNA library kit (Illumina) following the manufacturer’s protocol. The DNA library was quantitated using Qubit dsDNA assay kit (Life Technologies). Library DNA (4 nM) was loaded into a MiSeq Nano Flow Cell (300 cycles, Illumina) and sequenced with the Illumina MiSeq sequencing platform (Illumina). Three individual rounds of sequencing were performed and results combined to generate a consensus sequence for both BoHV-4 isolates. The BoHV-4 genome sequences were assembled with Ray [28], and the repeats were resolved using Celera [29] and Cap3 [30] software’s. Open reading frames (ORFs) for BoHV-4 isolates SD16-38 and SD16-49 were inferred and annotated using Geneious Prime software based on the genomic sequences of BoHV-4 strains 66-p-377 and FMV09 (GenBank accession numbers AF318573 and KC999113.1).
Animal study design
Two separate experiments were conducted to investigate the pathogenesis of contemporary BoHV-4 isolates. In experiment 1 four (n = 4) five month-old calves, negative for BoHV-4 neutralizing antibodies were used. After transport, animals were acclimated for 14 days and inoculated intranasally (IN) with BoHV-4 isolate SD16-49 (titer 106.3 TCID50/mL - 5 ml in each nostril). Animals were monitored daily for respiratory and systemic clinical signs. Rectal temperature was checked and recorded daily. Nasal swabs and blood samples were collected daily up to day 14 post-inoculation (pi). All procedures of animal handling and experimentation were conducted under veterinary supervision and according to recommendations by the Brazilian Committee of Animal Experimentation (approved by Institutional Ethics Committee UFSM - CEUA/UFSM protocol number 034/2014).
In experiment 2 eighteen 3-week-old colostrum-deprived Holstein bull calves negative for BoHV-4 neutralizing antibodies were allocated in three groups [31]. Following seven days of acclimation, 10 calves were inoculated with a suspension of BoHV-4 isolate SD16-38 (106.0 TCID50//mL) via IN instilation (5 ml in each nostril). Four calves were mock-inoculated with MEM (5 ml in each nostril) and kept as controls and the last four calves served as non-inoculated contact animals that were comingled with the inoculated animals starting at day 3 pi to evaluate potential virus transmission. Nasal swabs and blood samples were collected on days -1, 0, 3, 5, 7 and 10 pi. Two control calves and four inoculated calves were euthanized on days 5 pi and 10 pi. The four contact calves were euthanized on day 10 pi. The two remaining inoculated calves were euthanized on day 35 pi. Additional serum sampling for neutralizing antibody titration was performed on day 35 pi for the two inoculated calves. During the course of the study, animals were monitored daily and scored for clinical signs using Calf Health Scorer system developed at the University of Wisconsin School of Veterinary. Body temperature was constantly monitored using ruminal probes as previously described [31]. Euthanasia was performed with a pentobarbital sodium injection (Fatal Plus, Vortech Pharmaceutical Ltd., Dearborn, MI). The animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all procedures were approved by the Institutional Animal Care and Use Committee of the National Animal Disease Center (ARS-2016-572).
Sample collection and processing
Blood samples were collected by jugular venipuncture with Vacuette tubes containing clot activator (for serum), heparin (for PBMCs) or ethylenediaminetetraacetic (for complete blood cells count; CBC) (Greiner Bio-One, Monroe, NC). Serum samples were aliquoted and stored at −20 °C until tested. Blood samples collected with heparin were centrifuged at 1200 X g for 10 min and the buffy coat was transferred to a cryotube and stored at −80 °C until use. Tissues collected at necropsy from animals in experiment 2 included: nasal turbinate, thymus, tonsil, lung, spleen, olfactory bulb, trigeminal ganglia and lymph nodes (mandibular, retropharyngeal, tracheobronchial). Tissues were fixed by immersion in 10% neutral buffered formalin for RNAscope testing and placed in bags and frozen DNA extraction and PCR testing. Tissues were stored at −80 °C until use.
Viral DNA extraction and nested-PCR
Total DNA was extracted from serum, buffy coats and nasal swab samples using QIAamp® DNA (Qiagen) according to the manufacturer's instructions. In addition, total DNA was extracted from tissues collected in experiment 2, as follow: approximately 1 g of tissue was minced with sterile scalpel blade and mixed with 9 ml of MEM (1:10 w/v). Samples were homogenized with Stomacher and centrifuged 1200 X g for 10 min. DNA was extracted from tissue homogenates using QIAamp® DNA (Qiagen) according to the manufacturer's instructions. BoHV-4 DNA was detected using a previously described nPCR [24] with minor modifications. Briefly, nPCR targeting the BoHV-4 thymidine kinase region employed the following primers: 5’-GTTGGGCGTCCTGTATGGTAGC-3’ (primer 1), 5’-ATGTATGCCCAAAACTTATAATATGACCAG-3’ (primer 2), 5’-TTGATAGTGCGTTGTTGGGATGTGG-3’ (primer 3), and 5’-CACTGCCCG GTGGGAAATAGCA-3’ (primer 4). Primers 1 and 2 flanked a 567-bp fragment and the internal primers (primers 3 and 4) amplified a 260-bp product. Both PCR amplifications were performed with the same protocol using 50 μl reactions containing 25 μl of the Q5 hot start high-fidelity 2X master mix (New England Biolabs), 2.5 μl of each primer (final concentration of 0.5 μM), 19 μl nuclease-free water (Thermo-Fisher Scientific), and 1 μl of purified nucleic acid as template. The PCR conditions consisted of five initial amplification cycles at 94 °C for 45 s, 56 °C for 1 min, 72 °C for 1.5 min. Subsequently, an additional 25 cycles of 94 °C for 45 s, 51 °C for 1 min, and 72 °C for 1.5 min were performed followed by 72 °C for 5 min. PCR amplicons were analyzed by 1% agarose gel electrophoresis.
Virus isolation and quantification
Virus isolation in cell cultures was attempted from nasal swabs in experiment 1 (MDBK cells) and from serum, buffy coats and nasal swabs in experiment 2 (BT cells). Nasal swabs were placed in microtubes with 500 µl of MEM, vortexed for at least 30 s and 150 µl were inoculated in each well. For buffy coats and serum, 150 µl were inoculated in the wells and adsorption was performed for 1 h. Cells were washed with MEM twice and 500 µl of replacement media supplemented with 10% FBS were added to each well. Three passages were performed in 70–80% confluent cell monolayers. Plates were incubated at 37 °C with 5% CO2 and monitored daily for 6 days. Positive samples were quantified by limiting dilution and the titers calculated according to Reed and Muench method [32].
Virus neutralizing assays
Virus neutralization (VN) assays were performed with serum samples from experiment 2 (collected at day 35 pi). Briefly, serum dilutions from 1:2 to 1:512 were incubated with 200 TCID50 of the homologous virus (isolate SD16-38) during 90 min at 37 °C, followed by addition of a suspension of BT cells and incubation for 7 days at 37 °C in 5% CO2. Tests were read under light microscope for the presence of cytopathic effect. Neutralizing antibody titers were determined as the reciprocal of the highest serum dilution capable of completely inhibiting BoHV-4 replication.
RNAscope® in situ hybridization
RNAscope® in situ hybridization assay was used to investigate the presence of viral nucleic acid in tissues collected during experiment 2 from the animals necropsied on day 35 pi. Tissue sections were fixed by immersion in 10% neutral buffered formalin for 24 - 36 h, transferred to 70% ethyl alcohol until processed by standard paraffin-embedment techniques, cut in 5 μm sections for in situ viral labeling. Sequence from BoHV-4 isolate SD16-38 was used to design gene-specific oligonucleotide target probes per the manufacture’s recommendation. Labeling of viral nucleic acid using proprietary probes designed specifically to the BoHV-4 identification (Advanced Cell Diagnostics, Hayward, CA, USA) was performed according to manufacturer’s instructions for RNAscope® 2.0. Positive and negative control probes were used for verification of specificity of the assay. The positive control probe consisted of a proprietary probe for Bos taurus ubiquitin C (UBC; Cat # 464851), while the negative control probe targeted dapB of Bacillus subtilis (Cat # 312038).
Upon completion of the RNAscope® 2.0 assay and drying for approximately 15 min, slides were cover slipped using mounting media (EcoMount, Biocare Medical, Concord, CA, USA). Slides were scanned at 40X magnification and digitized using the Aperio ScanScope XT workstation (Aperio Technology, Inc., Vista, CA, USA). Digitized images were visualized using image analysis software (HALO™, Indica Labs, Inc., Corrales, NM) to describe BoHV-4 labeling patterns and compare to histological observations.