BOVINE RESPIRATORY DISEASES: MOLECULAR EPIDEMIOLOGY OF VIRAL INFECTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1000248
• Project No.: OKL02873
• Project Start Date: Oct 1, 2013
• Project End Date: Dec 31, 2015

Project Director
Fulton, RO.

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
Veterinary Medicine

Non Technical Summary
Viral infections are important contributors to bovine respiratory disease (BRD) in cattle. Viruses have devised ways to navigate around the immune response to infection and response to vaccination by changing their genetic makeup and antigens involved in immunity. Like human influenza these viruses by their mutation changes may be "emerging or reemerging "pathogens. It is important to monitor for the "new "viruses to assist the diagnostic laboratories to effectively identify the causative agents in BRD. Also these "new viruses" may not be susceptible to the vaccine induced immunity in cattle. For example the vaccines for bovine herpesvirus-1 (BoHV1) were developed and licensed in 1957. Similarly the vaccines in use today were developed for parainfluenza-3 virus (PI3V) and bovine viral diarrhea viruses (BVDV) over 20- 4o years ago. Thus this study will aid diagnostic labs, animal health companies, and the USDA APHIS Center for Veterinary Biologics for dealing with "new" BRD viruses. Another example of an emerging virus for BRD is the bovine coronavirus (BoCV). Our studies have found the current BoCV in Oklahoma is different from the enteric vaccine.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1060	50%
311	3310	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1060 - Biology (whole systems); 1101 - Virology;

Keywords

molecular epidemiology

bovine viruses

bovine respiratory diseases

genomics

viral sequencing

Goals / Objectives
Objective 1: Surveillance for bovine respiratory disease (BRD) viruses in cattle. Objective 2: Molecular characterization of BRD viruses including BoHV-1, BVDV, PI3V, BRSV, and BoCV from current studies and archived viral collection in CVHS. Objective 3: Differentiate MLV strains from field isolates Objective 4: Determine antigenic properties of "new "strains by in vitro and animal studies to determine immune response.

Project Methods
Procedures; Objective 1: Surveillance for BRD viruses in cattle. We will obtain samples including nasal swabs and serums from calves immediately upon arrival and after processing using the groups of calves at the Willard Sparks Beef Research Center (WSBRC). This approach has been used in prior studies as illustrated in our publication on BoCV (8). Nasal swabs and on occasion, lung washing fluids will be obtained on day 0 and weekly through day 14. We use sentinel calves representing a sample size of 15-20 animals. In addition serums are collected at day 0 and at 28-42 days after arrival. We will also collect samples from sick calves through day 14.We anticipate at least two groups yearly of 140-160 calves that are from auction markets in Oklahoma or Southeast US. Our experience has found evidence of BRD viruses in the first 14 days by viral isolation and serology (rising antibody titers from acute to convalescent serums. We have recovered BoHV-1, BVDV, and BoCV in these calves. We have access to commercial feedlots in Oklahoma, Kansas, and Texas whereby we can obtain respiratory tract samples and paired serums to monitor for BRD viruses. We anticipate at least two such studies yearly. The nasal swabs and lung washing fluids will be tested for viruses by inoculation of MDBK monolayer cultures for BoHV-1, PI3V, BVDV, and BRSV. (9) The BoCV will be detected by inoculation of HRT (human rectal tumor cell) monolayers, a highly sensitive cell line for BoCV. (8) In addition the serums, acute and convalescent will be tested for neutralizing antibodies to BoHV-1, BVDV, PI3V, BRSV, and BoCV by virus neutralization tests in cell culture (8, 17). Objective 2: Molecular characterization of BRD viruses including BoHV-1, BVDV, PI3V, BRSV, and BoCV from current studies and archived viral collection in CVHS. The genomic characterization of the viruses will utilize published protocols for BVDV (9) and for BoHV-1 (14). The BVDV typing is by sequencing of the 5'-UTR region and the BoHV-1 uses the SNPs pattern as compared using the primers to differentiate MLV strains and field strains different from the MLV strains. BoCV will be analyzed for genetic patterns into clades (genetic based groups) based on sequencing of a region of the envelope S region (11). The genetic characterization of the PI3V and BRSV will use the collaboration of Drs. Julia Ridpath and John Neill at the USDA ARS NADC, Ames, IA. To date we are not aware of the genome profile of PI3V and BRSV. The USDA laboratory has done recent work on the whole genome sequencing of our BoCV and this technology will be applied to the PI3v and BRSV. The procedure as described by Dr. Neill (unpublished) includes the following: Ion torrent sequencing will be conducted by constructing cDNA libraries of individual viruses using barcoded random priming of both first and second strain syntheses. After double-stranded cDNA synthesis, the DNA will be amplified using barcode-specific PCR primers. Individual virus libraries will be pooled, ion torrent adaptors ligated, and 300- 400 bp DNA isolated using a Pippin Prep. The size fractionated DNA will be sequenced using the standard Ion Torrent protocol and chemistry. Sequences will be demultiplexed and assembled using the Lasergene 10 Ngen template assisted assembly. This procedure has been used to further characterize for our BoCV using the whole genome sequencing. Objective 3: Differentiate MLV strains from field isolates: The BoHV-1 strains will be differentiated using the SNPs patterns for MLV vaccines and the PCR primers (14). The PCR products with these primers will be sequenced and the sequences compared to the MLV vaccine strains. Those not in the sequences will be considered as field strains unrelated to MLV strains. These procedures have been used to identify field strains of BoHV-1 (14) using BoHV-1 from BRD cases in cattle recently vaccinated with MLV strains. We have over 40 BoHV-1 in our archived collection of BoHV-1 which have not been characterized and we will use these in our study. The PI3V and the BRSV vaccine strains will be compared using the procedures described in Objective 2. Commercial vaccines will be obtained and the MLV strains will be characterized. Using commercial vaccines, the strains can be identified using viral sequencing. Such studies were used in a recent study of a 2012 Sparks BRD outbreak whereby commercial vaccine was obtained and the BVDV1a strain sequenced. Based on limited information the PI3V vaccine stocks and BRSV vaccine strains may be limited to the SF-4 strain for PI3V and the 375 strain for BRSV. (10) In our archived collection we have 5 PI3V strain which have not been characterized and also 5 BRSV isolates that will be used in the study. The BVDV strains in MLV vaccines utilize the sequencing of the 5'-UTR of the viral genome. In our archived viral collection we have over 40 BVDV strains which have not been sequenced and will be used in this study. The table below illustrates the vaccine strains of BVDV in the USDA licensed vaccines in the US. MLV Company Strain Genotype Biotype Express 5 BI Vetmedica Singer 296 1a CP 2a CP Pyramid 5 BI Vetmedica Singer 5912 1a CP 2a CP BoviShield 4 Zoetis NADL 1a CP BoviShield Gold 5 Zoetis NADL 53637 1aCP 2a CP Titanium 5 AgriLabs C24V 296 1a CP 2a CP Vista 5/ Onset Merck Singer 125A 1a CP 2a CP BRD Shield Novartis GL760 NAH 1024 1a NCP 2a NCP In summary we have an archived collection of BoHV-1, BVDV, PI3V, BRSV and BoCV which will be used in this study; these are in addition to the viruses to be obtained from proposed studies at WSBRC and cooperating feedlots. Objective 4: Determine antigenic properties of "new "strains by in vitro and animal studies to determine immune response. Selected viruses representing unique genetic c distinct viruses representing each of the groups of BoHV-1, PI3v, BRSV, BVDV and BoCV will be compared for antigenic differences using virus neutralization tests (VNT) in cell culture.(17) Also there will be animal studies performed to determine the immune response in the calves using acute and convalescent serums in the vaccinated calves. There will be a minimum of 3 genetically distinct viruses for each of the BoHV-1, PI3V, BVDV, BRSV, and BoCV used in the study. Calves will receive experimental vaccines containing 105 TCID50 given by the subcutaneous route. There will be four calves each for each virus. The immune response to the "new" virus will be compared to those antibody levels of current MLV vaccinated calves. Commercial vaccines with MLV strains will be included. We have archived serums from calves that have received MLV vaccines and these will be tested again using the "new "strains. It is expected that the "new" strains will induced different levels of antibodies.

Progress 10/01/13 to 12/31/15

Outputs
Target Audience:The audience for this research included researchers of bovine respiratory diseases (BRD), diagnosticians including virologists, pathologists, epidemiologists, veterinary clinicians, and cattle producers. Also included are extension faculty, and animal health companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supported a pre-veterinary student who was an honor student in biochemistry and an early admitted student to the Oklahoma State University class of 2019. How have the results been disseminated to communities of interest?Results of this project have been published or presented at scientific meetings also to seminars to animal health companies. This project will be terminated as the principal investigator will be retiring Oct 1, 2015. What do you plan to do during the next reporting period to accomplish the goals?This project is being terminated

Impacts
What was accomplished under these goals? 1. Bovine herpesvirus-1, bovine viral diarrhea viruses, parainfluenza-3 virus, bovine respiratory syncytial virus and bovine coronaviruses were isolated from cattle with respiratory disease. 2. Bovine coronaviruses were characterized by sequencing a region of the envelope S gene. The bovine herpesvirus-1 isolates were found to be genomically distinct by whole genome sequencing and identifying single nucleotide polymorphisms (SNPs). The MLV vaccine strains were separated by distinct SNPs pattern. MLV BoHV-1 strains could be differentiated from field strains. BoHV-1 isolates from aborted fetuses whose dams received MLV vaccines were found to be identical to the MLV strain given to the pregnant dam. BoHV-1.2b, the genital form of IBR, was found in calves with BRD. 3. Bovine coronaviruses were found to be clade 2 and 3 based on genetic differences and antigenic differences from clade 1. The clade 1 represented the BoCV vaccine strain and the neonatal calf diarrhea strain. BoCV clade 2 and 3 were found in calves with BRD that were commingled and mixed source auction calves. BoCV clade 2 was found in postweaned calves with fatal acute hemorrhagic colitis. 4. Bovine viral diarrhea viruses were found in multiple forms of fetal disease including abortions and persistently infected newborn calves. BVDV1b was the predominant subtype in these disease forms, BVDV2a was found in an aborted fetuses with congenital anomalies resembling an Angus genetic defect of the musculoskeletal system.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Fulton,R.W.: Host Response to Bovine Viral Diarrhea Virus and Interactions with Infectious Agents in the Feedlot and Breeding Herd. Biologicals, 41:31-38, 2013.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Fulton, R.W., Ridpath, J.F., Burge, L.J.: Bovine Coronaviruses From the Respiratory Tract: Antigenic and Genetic Diversity. Vaccine, 31: 886-892,2013.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: dOffay, J.M., Fulton,R.W., Eberle, R.: Complete Genome Sequence of the NVSL BoHV-1.1 Cooper Reference Strain. Archives of Virology, 158: 1109-1113, 2013.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Fulton, R.W., dOffay, J.M., Eberle, R.: Bovine Herpesvirus-1: Comparison and Differentiation of Vaccine and Field Strains Based on Genomic Sequence Variation. Vaccine, 31:1471-1479, 2013 ..
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fulton, R.W., Rezabek, G.B., Grant, R., Ridpath. J.F., Burge.L.J.: Diverse Outcomes of Bovine Viral Diarrhea Virus Infections in a Herd Naturally Infected During Pregnancy- a Case Study.. Bovine Practitioner, 48: 95-98, 2014.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fulton, R.W., Herd, H.R., Sorensen, N.J., Confer, A.W., Ritchey, J.W., Ridpath, J.F., Burge.L.J.: Enteric Disease in Postweaned Beef Calves Associated with Bovine Coronavirus Clade 2. Journal of Veterinary Disease Investigation, 27: 97-101, 2015.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fulton, R.W., dOffay J.M., Eberle,R., Moeller, R.B., Van Campen, H., OToole, D., Chase, C., Miller. M.M., Sprowls, R., Nydam, D.V.: Bovine Herpesvirus-1: Evaluation of Genetic Diversity of Subtypes Derived from Field Strains of Varied Clinical Syndromes and Their Relationship to Vaccine Strains. Vaccine, 31: 549-558, 2015.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fulton, R.W.: Impact of Species and Subgenotypes of Bovine Viral Diarrhea Virus on Control by Vaccination. Animal Health Research Reviews, 16: 40-54, 2015.
• Type: Book Chapters Status: Published Year Published: 2014 Citation: Large Animal Internal Medicine: Fifth Edition, B.P. Smith, Editor, Dr. V. Cortese, Section Editor, Vaccines. Bovine herpesvirus-1 Vaccines for Cattle in Bovine Respiratory Disease Vaccines Chapter, pp. 1471-1475. 2014
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fulton, R.W.,  Bovine Respiratory Disease: Diagnosis and Prevention, European Buiatrics Forum 2013, sponsored by French Buiatrics Association, Marseille, France, November 27-29,2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fulton, R.W.: Infectious Bovine Rhinotracheitis and Its Effects on Reproduction- Evaluation of Current Vaccines. Western Veterinary Conference, Las Vegas, NV. February 20, 2013.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The audiences for the research in this study include: researchers in bovine respiratory diseases (BRD), diagnosticiana such as virologists and pathologists working with clinicians and producers on BRD cases, farmers and ranchers with cattle, extension faculty in livestock, and animal health companies. Changes/Problems: I anticipate no problems in the continued research. What opportunities for training and professional development has the project provided? This study included a pre veterinary student who is an early admit student to the OSU veterinary school. She is also an honors student in Biochemistry with a potential career in research. How have the results been disseminated to communities of interest? The results have been published or presented at scientific meetings. The results have been presented to animal health companies. What do you plan to do during the next reporting period to accomplish the goals? Continue viral isolation in cell culture from cases of BRD and to characterize the isolates by genomic sequencing.

Impacts
What was accomplished under these goals? 1. Bovine herpesvirus-1 (BoHV-1) was isolated for cattle with BRD as well as bovine coronaviruses (BoCV) 2. The BoCV viruse were characterized by sequencing of a region of the spike protein of the envelope. The BoHV 1 isolates were found to be genetically distinct from the vaccine viruses in some cases. However the isolates from aborted fetuses from MLV vaccinated dams were identical to MLV vaccine strain. BoHV 1.2b was isolated from cattle with BRD. 3. The BoHV 1.1 strains from selected cases were related to MLV vaccine strain. 4. The BoCV isolates fBoCV clade 2) rom BRD cases were antigenically distinct from the BoCV 1 clade.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fulton, R.W., Rezabek, G.B., Grant, R., Ridpath. J.F., Burge.L.J.: Diverse Outcomes of Bovine Viral Diarrhea Virus Infections in a Herd Naturally Infected During Pregnancy- a Case Study.. Bovine Practitioner, 48: 95-98, 2014.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fulton, R.W., Herd, H.R., Sorensen, N.J., Confer, A.W., Ritchey, J.W., Ridpath, J.F., Burge.L.J.: Enteric Disease in Postweaned Beef Calves Associated with Bovine Coronavirus Clade 2. Journal of Veterinary Disease Investigation, 26: 2014. In Press.
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Fulton, R.W., dOffay J.M., Eberle,R., Moeller, R.B., Van Campen, H., OToole, D., Chase, C., Miller. M.M., Sprowls, R., Nydam, D.V.: Bovine Herpesvirus-1: Evaluation of Genetic Diversity of Subtypes Derived from Field Strains of Varied Clinical Syndromes and Their Relationship to Vaccine Strains. Vaccine, Accepted for publication. November 16,2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fulton, R.W., Ridpath, J.F., Neill, J., Burge, L.J., Wilson, B., Maxwell, C., Step, D.L: Bovine Viral Diarrhea Virus (BVDV) in Postweaned Calves in a Feedlot after Vaccination and from Fatal Respiratory Cases: Isolation and Differentiation of Modified Live Viral (MLV) BVDV and Field Strains. 56th Annual Meeting of AAVLD, October 17-21,2013, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brown, J., Woolums, A.,Jones, L,McKinnon, G., Fulton, R.W., Ridpath, J.: Investigation Into an Outbreak of Respiratory Disease in Nursing Calves in a South Georgia Beef Herd. Bovine Respiratory Disease Symposium (BRDS) 2014. Denver , CO. July 30-31,2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fulton, R.W.: Bovine Coronaviruses: Respiratory and Digestive Tract Infections/Disease in Post Weened Beef Calves. Academy of Veterinary Consultants Meeting. Augest 1-2,2014. Denver, CO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Herd, H.R., Ritchey, J.W., Fulton, R.W., Ridpath, J.F.: Coronavirus-Associated Hemorrhagic Colitis in Two Young Adult Beef Cattle in Oklahoma. 57th Annual Meeting of AAVLD, October 26=22-21,2014. Kansas City, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fulton, R.W.: Impact of Species and Subgenotypes of BVDV on Control by Vaccination. Joint U.S. BVDV/ESVV Pestivirus Symposium. October 14-15,2014. Kansas City, MO.