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VETMIC-6587; No. of Pages 5 Veterinary Microbiology xxx (2014) xxx–xxx

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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Short communication

Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2 Naomi S. Taus a,*, Donal O’Toole b, David R. Herndon a, Cristina W. Cunha c, Janet V. Warg d, Bruce S. Seal e, Angela Brooking c,1, Hong Li a a

USDA-ARS-ADRU, Washington State University, Pullman, WA 99164-6630, USA Wyoming State Veterinary Laboratory, University of Wyoming, Laramie, WY 82070, USA Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA d National Veterinary Services Laboratories, USDA, Ames, IA 50010, USA e Poultry Microbiological Safety Research Unit, USDA-ARS, Athens, GA 30605, USA b c



Article history: Received 5 December 2013 Received in revised form 25 March 2014 Accepted 1 April 2014

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison (Bison bison) throughout North America. No vaccine exists to protect animals from disease. Since OvHV-2 has not been propagated in vitro, one strategy to develop a modified live vaccine is to use a closely related, non-pathogenic member of the malignant catarrhal fever virus family as a vector expressing potentially protective OvHV-2 epitopes. To date, no controlled experimental challenge studies with alcelaphine herpesvirus 2 (AlHV-2) derived from topi (Damaliscus lunatus jimela) have been reported The unique or light DNA segment of the AlHV-2 genome was sequenced and annotated and the virus was tested for its ability to infect and induce disease in American bison. Yearling bison were inoculated intranasally (n = 4) or intramuscularly (n = 3) with 2  104.7 TCID50 of AlHV-2, and monitored for infection and the development of disease. Six inoculated bison became infected with AlHV-2. Two of the six animals developed clinical signs and had gross and histological lesions consistent with terminal MCF, which differed in distribution from those in bison with MCF due to OvHV-2. One other animal developed minor clinical signs and had gross and histological pulmonary lesions consistent with early (pre-clinical) stages of MCF. Unmodified low cell culture passage AlHV-2 derived from topi is an unsuitable vaccine vector for the prevention of MCF. However, the annotated genome might be useful in identifying genes which could be deleted to potentially attenuate the virus for bison. Published by Elsevier B.V.

Keywords: Malignant catarrhal fever American bison OvHV-2 AlHV-2

1. Introduction Malignant catarrhal fever (MCF) is a generally fatal disease, caused by some viruses in the genus Macavirus,

* Corresponding author. Tel.: +1 509 335 6318; fax: +1 509 335 8328. E-mail address: [email protected] (N.S. Taus). 1 Current address: Mill Creek Veterinary Hospital, Walla Walla, Washington 99362, USA.

subfamily Gammaherpesvirinae, affecting many ruminant species including domestic cattle, most cervid species, and North American bison (Bison bison). Deaths due to MCF caused by ovine herpesvirus 2 (OvHV-2), which is carried by most domestic sheep (Ovis aries), result in significant losses in the ranched bison (B. bison) industry in the United States and Canada. The development of an effective vaccine against MCF should improve control of the disease in susceptible species. An impediment to vaccine development is the inability to propagate OvHV-2 in vitro. An

http://dx.doi.org/10.1016/j.vetmic.2014.04.003 0378-1135/Published by Elsevier B.V.

Please cite this article in press as: Taus, N.S., et al., Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.04.003

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alternative approach is to use a closely related, nonpathogenic MCF virus as a vector for genes encoding key antigens of OvHV-2. Alcelaphine herpesvirus 2 (AlHV-2) isolated from topi (Damaliscus lunatus korrigum) is considered non-pathogenic based on limited experimental studies involving parenteral inoculation of cattle and rabbits (Mushi et al., 1981). AlHV-2 derived from hartebeest (Alcelaphus buselaphus), another African antelope species, induced MCF when inoculated into cattle and rabbits (Reid and Rowe, 1973) and an AlHV-2-like virus thought to have originated in hartebeest caused MCF in Barbary red deer (Klieforth et al., 2002). It may be appropriate to consider AlHV-2 derived from topi and hartebeest as two distinct members of the Macavirus genus, and to use the designations topiAlHV-2 and hartebeest-AlHV-2 until their relationship is clarified. Topi-AlHV-2 has been propagated in embryonic topi and fetal aoudad (Ammotragus lervia) cell cultures (Mushi et al., 1981; Seal et al., 1989), and could be manipulated to generate a recombinant virus carrying desired OvHV-2 genes. The infectivity of the virus for bison was determined and the unique or light DNA segment of the topi-AlHV-2 genome was sequenced and annotated. 2. Materials and methods 2.1. Virus culture and genome sequencing The AlHV-2 isolate 840412, originally isolated from a topi (Damaliscus lunatus ssp. jimela) at the Zoological Society of San Diego Zoo (Seal et al., 1989), was propagated in fetal mouflon sheep (Ovis aries orientalis) kidney cells (FMSK) maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin (100 units/ml), streptomycin (100 mg/ml), and Fungizone (250 ng/ml). AlHV-2 inoculum was prepared by infecting FMSK cells, lysing cells by three freeze-thaw cycles once the cytopathic effect reached 80%, and clarifying the lysate by centrifugation for 25 min at 1159  g. Aliquots were stored at 80 8C until use. The tissue culture infectious dose (TCID50) of the inoculum was determined on FMSK cells. For sequencing of the viral genome, culture supernatant from infected FMSK cells was harvested when cytopathic effect reached 80%. Virions were collected by ultracentrifugation through a 35% sucrose cushion and further clarified by DNAse digestion (0.5 mg/ml; Roche) to remove unprotected viral and cellular DNA. Viral DNA was released by the addition of proteinase K (0.5 mg/ml; Sigma-Aldrich) and SDS (0.3%), extracted twice with phenol/chloroform/ isoamyl alcohol (24:24:1), and precipitated in 95% ethanol. The purified topi-AlHV-2 DNA was sheared with a Hydroshear (Genomic Solutions) and a shotgun library (4–5 kb) constructed using the pSMARTLCKan kit (Lucigen) according to the manufacturer’s instructions. Clones (768) were sequenced in the forward and reverse directions using the BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 3130xl Genetic Analyzer (both from Applied Biosystems/Life Technologies). Sequence assembly was performed with PHRAP (www.phrap.org, Ewing and Green, 1998) and resulting contigs compared to the complete

sequence of the AlHV-1 strain C500 as a scaffold to simplify orientation and link contigs. Annotation was performed manually and submitted to GenBank (KF274499). 2.2. Animal inoculation and sampling Seven yearling bison (five male; two female), were purchased from a commercial producer and maintained at the animal facility at the University of Wyoming, Laramie, WY. The study was done in accordance with a universityapproved animal care and use protocol. Before they were purchased, the bison were screened for the presence of OvHV-2 DNA using a semi-nested PCR assay (Li et al., 2004) and for MCF viral (MCFV) antibodies using a competitive inhibition ELISA (cELISA) (Li et al., 2001). None of the 7 had detectable OvHV-2 DNA or MCFV antibodies. Because the AlHV-2 stock was contaminated with a bovine viral diarrhea virus (BVDV) determined to be a type 1b genotype (data not shown), attempts were made to eliminate BVDV from small aliquots of the stock using goat anti-BVDV (type 1 and type 2) neutralizing antibodies (kindly provided by Dr. Chungwon Chung, VMRD, Pullman, WA). This approach did not eliminate viral contamination so it was decided to use low passage (p7) untreated stock for the study. To mitigate confounding effects of BVDV infection, bison were immunized against BVDV on three occasions before inoculation with topi-AlHV-2, as described below. The seven bison were vaccinated on three occasions against BVDV using a polyvalent commercial vaccine (Pyramid1 4 + Presponse1 SQ; Boehringer Ingelheim Vetmedica, Inc., Fort Dodge, IA 50501). The BVDV component in the vaccine consisted of modified live Singer strain (1a genotype; cytopathic biotype) (Fulton, 2005). The other biological components in the vaccine were modified live bovine parainfluenza 3, bovine respiratory syncytial virus, and a Mannheimia haemolytica toxoid. The third and final vaccination was given four weeks before AlHV-2 inoculation. Four bison were inoculated via intranasal nebulization (IN), as described previously for OvHV-2 (Li et al., 2004), with 2  104.7 TCID50 of topi-AlHV-2. Three other bison were inoculated with the same dose of virus intramuscularly (IM) in the thigh. Blood was collected weekly and assayed for topi-AlHV-2 DNA and MCFV antibodies. One of the IN inoculated bison died from intercurrent disease before any data could be obtained. A complete set of tissues was collected from all animals at the time of necropsy and fixed in 10% buffered formalin. Fixed tissues were embedded in paraffin and processed routinely for histological examination. 2.3. Nucleic acid and antibody analysis DNA was extracted from peripheral blood leukocytes (PBL) and from tissues [liver, tracheobronchial (TB) lymph node, spleen, lung and urinary bladder] collected post-mortem using the Fast DNA kit (QBiogene) according to the manufacturer’s directions. A 75 bp fragment of the AlHV-2 A4.5 gene was amplified and detected using the CFX96 Real-Time PCR Detection System (Bio-Rad).

Please cite this article in press as: Taus, N.S., et al., Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.04.003

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Reactions were performed using 250 nM of each forward (AlHV-2-4.5 F2, 50 aatccagtgcctgtaaaac) and reverse (AlHV-2-4.5 R1, 50 cgcagattcctccagt) primers; 100 ng of total DNA, and 1 SsoFast EvaGreen1 Supermix (BioRad). Serial dilutions of a plasmid containing the amplicon sequence (1  108 to 1  101 copies per reaction) were used in each run to construct a standard curve. The assay’s limit of detection was one topi-AlHV-2 genome copy, with a dynamic range for quantification of 101 to 108 genome copies per 100 ng of total DNA. The cycling protocol was 95 8C/30 s followed by 40 cycles of 95 8C/15 s and 60 8C/10 s with a plate read after each cycle. To confirm amplification specificity, melting curve analysis (0.5 8C increments and readings at each 4 s from 65 to 95 8C) were performed following each PCR run. Additionally, the PCR did not amplify DNA from OvHV-2, AlHV-1, caprine herpesvirus 2, caprine herpesvirus 3 (MCFV carried by white-tailed deer), Ibex-MCFV, and bovine lymphotropic herpesvirus. The qPCR results were analyzed using the CFX Manager Software (Bio-Rad) and reported as topi-AlHV-2 genome copies/100 ng DNA based on the standard curve. The cELISA was used to detect MCFV antibodies in plasma (Li et al., 2001). Inhibition 25% was considered positive for the presence of MCFV antibodies. Plasma collected from all bison on day 0 post infection (PI) and at various times PI (Table 1) was submitted to the Washington State Animal Disease Diagnostic Laboratory, Pullman, WA to test for neutralizing antibody against BVDV. Plasma and liver samples from bison nos. 1, 3 and 5 were also submitted to test for BVDV RNA using a real time PCR assay. 2.4. Immunohistochemistry Immunohistochemistry for evidence of BVDV antigen involved using a commercially available antibody (IDEXX) (Haines et al., 1992), appropriate positive controls, and a published procedure (Cornish et al., 2005). At least three tissues with histological lesions from each bison were assessed. Tissue from a steer with virologically confirmed mucosal disease was used as a positive control.


3. Results 3.1. Topi-AlHV-2 infection of bison The topi-AlHV-2 infection status of inoculated bison was monitored using cELISA and qPCR (Table 1). Animals seropositive for MCFV antibodies or with tissues positive for topi-AlHV-2 DNA were considered infected. The three IM inoculated bison (animals nos. 4–6) developed detectable MCFV antibodies by 6 (nos. 4 and 5) and 20 (no. 6) days post inoculation (DPI). Bison no. 5 presented with oculonasal discharge two days before euthanasia on 23 DPI, and was in sternal recumbency on the day of euthanasia at 25 DPI. A complete necropsy was performed and six tissues were examined for topi-AlHV-2 DNA using qPCR. Viral DNA, reported as genome copies/100 ng total DNA, was detected in liver (34 copies/100 ng), TB lymph node (1890 copies/100 ng), spleen (

Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2.

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison (Bison bison) throughout North Am...
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