Abstract
Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen capable of causing disease affecting multiple body systems. Previous studies have shown 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) effectively prevents BVDV infection in cell culture. The aim of this project was to assess the efficacy of DB772 for the prevention of acute BVDV infection. Four calves seronegative to BVDV were treated with DB772 and another 4 calves were treated with diluent only on the same dosing schedule. Each calf was subsequently challenged intranasally with BVDV. Virus was isolated consistently from untreated calves on days 4 to 8, while treated calves remained negative by virus isolation during this period. Azotemia was exhibited by all treated calves on day 4 resulting in the euthanasia of 1 calf on day 10 and the death of another on day 13. Virus was isolated from the 2 remaining treated calves on day 14 or 21. On day 21, both remaining treated calves and all 4 untreated calves had anti-BVDV antibody titers > 1:2048. This pilot study indicates that DB772 temporarily prevented acute disease due to BVDV, but carries a significant concern of renal toxicity.
Résumé
Le virus de la diarrhée virale bovine (BVDV) est un agent pathogène bovin largement répandu capable de causer une pathologie affectant de nombreux systèmes organiques. Des études antérieures ont démontré que le 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phényl] dihydrochlorure furan (DB772) empêche efficacement l’infection par le BVDV en culture cellulaire. L’objectif de ce projet était d’évaluer l’efficacité du DB772 à prévenir une infection aiguë par le BVDV. Quatre veaux séronégatifs pour le BVDV ont été traités avec du DB772 et quatre autres veaux ont été traités avec uniquement du diluant en suivant la même cédule de traitement. Chaque veau a par la suite été infecté par voie intranasale avec du BVDV. Du virus a été isolé de manière constante à partir des veaux non-traités des jours 4 à 8, alors que les veaux traités sont demeurés négatifs pour l’isolement viral durant cette période. Une azotémie a été notée chez tous les veaux traités au jour 4 ce qui entraina l’euthanasie d’un veau au jour 10 et le décès d’un autre au jour 13. Du virus fut isolé à partir des deux veaux traités restant au jour 14 ou 21. Au jour 21, les deux veaux traités restant et les quatre veaux non-traités avaient des titres d’anticorps anti-BVDV > 1:2048. Cette étude pilote montre que le DB772 a empêché temporairement une maladie aiguë due au BVDV, mais laisse entrevoir de sérieuses inquiétudes quant à sa toxicité rénale.
(Traduit par Docteur Serge Messier)
Introduction
Bovine viral diarrhea virus (BVDV) is the prototypical virus of the Pestivirus genus of the Flaviviridae family (1). Clinical disease caused by BVDV infection is a source of significant economic losses in cattle worldwide (2). Acute disease caused by BVDV can cause several different clinical syndromes, which are influenced by viral strain and several host factors (e.g., age, immune status, stage of gestation) (3). The clinical effects of BVDV are manifested in the respiratory, gastrointestinal, reproductive, cardiovascular, lymphatic, immune, integumentary, or central nervous systems of affected cattle (4). Animals are usually infected through the respiratory or gastrointestinal mucosa, but the virus can also be transmitted transplacentally or via artificial insemination with contaminated semen (1). Subsequent primary viremia results in widespread dissemination of the virus. Acute BVDV infection marked by severe thrombocytopenia and hemorrhage has also been described (5). Other than maintaining a strong biosecurity program, including identification and culling of persistently infected animals, current preventatives for BVDV infection are limited to killed or modified live vaccines.
Aromatic cationic compounds possess inhibitory action against several RNA viruses, including human immunodeficiency virus (6), rotavirus (7) and respiratory syncytial virus (8). Givens et al (9) used a similar novel compound, 2-(2-benzimidazolyl)-5- [4-(2-imidazolino) phenyl]furan dihydrochloride (DB772; MW = 410.28), to inhibit BVDV growth in cell culture while demonstrating the compound’s lack of cytotoxicity. The 99% endpoint for prevention of BVDV replication by DB772 was found to be 6 nM. Furthermore, DB772 has been shown to eliminate BVDV infection in contaminated bovine fetal fibroblast cells with a single passage in culture media supplemented with 4 μM of DB772 (10). Blastocyst development was not hindered by exposure to a closely related compound and heifers resulting from treated blastocysts displayed normal characteristics during puberty, breeding, gestation, and lactation (10,11). Administration of the compound to calves persistently infected with BVDV resulted in a decrease in viral concentration with no detectable negative side effects (12). However, administration of DB772 to calves as a means to prevent BVDV infection has not been studied. Therefore, the purpose of this pilot study was to initiate an evaluation of the antiviral activity of DB772 when administered to healthy calves subsequently challenged intranasally with BVDV.
Materials and methods
Animals and housing
Eight miniature Brahman-cross calves (4 heifers and 4 bulls) that were between 5 and 6 mo of age were used in this study. Five days prior to study initiation, the calves were weaned, weighed, and allowed to acclimate to the project housing until the study onset. Calves were allowed free choice access to a commercially available calf starter, grass hay, and clean water when introduced to the study housing and throughout the study period. Reconstituted oral electrolyte powder was offered daily to calves from day 9 to day 18. All calves were determined to be seronegative to BVDV-2 (≤ 1:4) by virus neutralization prior to study initiation. Likewise, BVDV was not isolated in serum, buffy coat, or nasal swab samples from any calf using the immunoperoxidase monolayer assay before commencement of treatment.
The calves weighed between 46.7 and 95.2 kg (mean: 72.4 kg, standard deviation: 18.2) at the study onset. The calves were stratified by weight into 4 strata (2 calves per strata) and assigned random numbers using computer software (Microsoft Excel; Microsoft, Redmond Washington, USA). In each strata, the calf with the higher random number was assigned to group A, the calf with the lower number to group B. Based on a coin toss, group A (3 males, 1 female) was designated the treatment group and group B (1 male, 3 females) was designated the control group.
The calves were housed in individual 1.9 meter square crates arranged 2 per room within a 9.3 meter square pen inside a humidity- and temperature-controlled room. Each room housed 1 calf from the treatment group and 1 calf from the untreated group. This study design was intended to minimize potential environmental bias between the treatment and control groups that may have been introduced by housing them in separate rooms. Rooms were cleaned and calves observed at least twice daily. During the daily cleaning period, calves were released into a communal area within each room to allow for cleaning of the individual crates.
Clinical monitoring
Physical examinations including rectal temperatures were done on all calves daily throughout the study period and a second daily temperature reading was obtained beginning on day 6 until day 12. Calves were considered febrile when rectal temperatures exceeded 39.3°C. Clinical scores for appearance, dehydration status, appetite, and fecal appearance were recorded daily. Appearance scores were based on attitude and behavior with a score of 0 being normal to 1.0 being defined as severe depression, recumbency and refusal to rise or move even with encouragement. Dehydration scores were based on capillary refill time, skin tent, and sunken eyes with a score of 0 being normal to 1.0 being defined as a capillary refill time exceeding 3 s, skin tent exceeding 10 s accompanied by sunken eyes and cold extremities. Appetite scores were based on severity and duration of anorexia with a score of 0 being normal, a score of 0.2 defined as a decreased appetite with limited interest in eating and a score of 0.4 defined as complete anorexia. Fecal scores were based on the severity of diarrhea and the presence or absence of fecal blood with a score of 0 being normal, a score of 0.2 indicating watery diarrhea and a score of 0.4 denoting blood in the feces. Humane endpoints for euthanasia based on clinical scores or physical examination findings were established prior to study onset and defined as a dehydration score of 1 (sunken eyes, severe loss of skin elasticity, cold extremities) at any time point or when appearance, appetite, dehydration, and fecal clinical scores summed to a total of 1.0 or greater for 2 consecutive days.
Treatment
The compound DB772 was synthesized in the laboratory of one of the authors (D.W. Boykin, Georgia State University, Atlanta, Georgia, USA). A 25 mg/mL solution of DB772 was made by dissolving the compound in polyethylene glycol 200 (PEG 200) (Mallinckrodt Baker, Phillipsburg, New Jersey, USA). After a 5 d adjustment period to the isolation facilities, jugular catheters were placed bilaterally in all 8 calves. The following day treatment with DB772 was initiated in the treatment group calves (A, B, C, D) and administration of diluent in the untreated group calves (E, F, G, H). Treatments in each group were administered intravenously via the right jugular catheter every 8 h for a total of 11 treatments over 4 d. Day 0 was defined as the day treatment was first initiated with the final treatment administered on day 3. The antiviral dose (12 mg/kg BW) was calculated to achieve a 4 μM concentration (1.6 μg/mL) in serum.
Virus
The virus used in these studies (courtesy of J. Ridpath, USDA National Animal Disease Center, Ames, Iowa, USA) was a noncytopathic BVDV-2 (strain 1373) originally isolated from a 1993 outbreak of clinically severe acute BVDV infection in Ontario, Canada (13). After administration of the second treatment on day 0, each calf was inoculated with 40 000 cell culture infectious doses (CCID50) of BVDV-2 (1373) by aerosol inoculation using a DeVilbiss aerosolizer. This selected dose of virus was 1 to 2 logs less than used in previous research (14,15) in an effort to slow the rapid course of severe disease, which can be seen with this strain (16).
Sample collection
Whole blood and serum samples were collected on days 0, 2, 4, 6, 8, 14, 21, and 28 via the left jugular catheter or by jugular venipuncture if no catheter was present. Nasal swab samples were collected on days 0, 4, 8, 14, 21, and 28. Serum was removed from clotted blood after centrifugation and processed immediately or refrigerated until processing. Buffy coat samples were obtained from whole blood samples, as described earlier (17), with the exception that samples were resuspended in 1 mL of minimum essential medium. Whole blood, serum, and nasal swabs were refrigerated for < 72 h before sample analysis. Whole blood and serum samples from each calf were submitted to the clinical pathology service at Auburn University Teaching Hospital for complete blood (cell) count (CBC) and serum biochemical profile (SBP) analysis on days 0, 4, 8, 14, 21, and 28. Analytes assessed by SBP evaluation included total protein, albumin, globulins, serum dehydrogenase, aspartate transaminase, gamma-glutamyl transpeptidase, total bilirubin, creatine kinase, urea nitrogen, creatinine, calcium, phosphorous, glucose, magnesium, bicarbonate, sodium, potassium, chloride, and iron.
Virus and antibody detection
Virus isolation was done on buffy coat, serum, and nasal swab samples by using the immunoperoxidase monolayer assay, as described elsewhere (17), with the exception that 1 mL aliquots of buffy coat sample were used and the procedure was done in 6-well (9.6 cm2) plates. Virus neutralization assays were done on serum samples collected on days 0, 7, 14, and 21, as described previously, except that serum was not initially diluted (18). The virus neutralization assays were done using BVDV2 (1373). Plates were incubated for 3 d at 38.5°C in a humidified atmosphere of 5% CO2 and air, and then underwent the immunoperoxidase monolayer assay procedure for detection of BVDV (18).
Results
Physical and hematologic findings
At study initiation, all calves were judged to be healthy based on a complete physical examination. Whole blood and serum samples submitted for CBC and SBP analysis on day 0 revealed no remarkable differences between individual calves or between groups. However, on day 4, all calves in the treated group had elevated serum creatinine concentrations ranging from 2.6 to 7.6 mg/dL [reference range (RR): 1.0 to 2.3 mg/dL] and elevated serum urea nitrogen (SUN) concentrations of 38 to 84 mg/dL (range: 6.9 to 16.8 mg/dL). The same analytes were within the reference range for all calves in the untreated group. On day 8, serum creatinine concentrations in calves A and D had returned to normal, but remained high in calves B and C (6.9 and 19.3 mg/dL, respectively). Likewise, SUN concentrations remained elevated in treated calves B, C, and D (80.7, 131.9, and 22.7 mg/dL, respectively) and were also slightly outside the reference range in untreated calves E and F (17.2 and 17.7 mg/dL, respectively). Azotemia resolved in calves E and F by day 14, but SUN remained mildly elevated in calf D (20.7 to 28.7 mg/dL) throughout the study.
Transient inappetence was seen in calf A on day 3 and mild inappetence and depression in calf C on day 4. Calves B and C exhibited decreased appetite beginning on day 3 that progressed to complete anorexia on days 11 and 8, respectively. No evidence of clinical dehydration was noted at any point during the study. Calf C was humanely euthanized on day 10 due to continued depression and inappetence. Intravenous fluid and electrolyte therapy was instituted for calf B beginning on day 11. Despite aggressive treatment, the calf died on day 13.
Tissues from calves B and C were examined by a board-certified veterinary pathologist and revealed multifocal tubulointerstitial nephritis with tubular epithelial degeneration, necrosis, and regeneration. The renal tubules contained variable numbers of sloughed cells and inflammatory cells and moderate amounts of eosinophilic fluid. Mild to moderate splenic lymphoid depletion was present in both calves.
On day 14, 2 untreated calves (E and H) developed loose stool that progressed to watery diarrhea over a 24 h period. Calf H exhibited complete anorexia and mild depression that continued for 3 d. No treatment was necessary beyond the provision of free-choice reconstituted oral electrolyte solution. Fecal scores returned to normal after 3 (calf E) to 7 d (calf H). Diarrhea was not present in the treated calves at any point during the study. Clinical scores for appearance were normal throughout the study period for all untreated calves.
The average lymphocyte count in treated calves on day 0 was 5306 × 103/μL (RR: 2500 to 7500 × 103/μL) with a standard deviation (SD) of 1654 and 5193 × 103/μL (SD = 1574) in untreated calves (Figure 1). All individual lymphocyte counts were within the reference range on day 0. The average lymphocyte count in untreated calves decreased 48% to 2703 × 103/μL (SD = 611) from day 0 to day 4 before increasing slightly to 2947 × 103/μL (SD = 983) on day 8. On day 4, calf E had a lymphocyte count of 1863 × 103/μL, which remained outside the reference range on day 8 at 2260 × 103/μL. Calf H had a low lymphocyte count on day 8 at 2134 × 103/μL and calf F had a count of 2373 on day 14. Lymphocyte counts of calf G were within the reference range at all time points. The average lymphocyte count in treated calves decreased to 4700 × 103/μL (SD = 1604) on day 4 and 4020 × 103/μL (SD = 634) on day 8, a decline of 11% and 24%, respectively, with no individual lymphocyte counts falling outside the reference range. The average lymphocyte count returned to within 90% of baseline on day 14 in the treated group and day 21 in the untreated group. Differences in average lymphocyte count were not statistically significant (P = 0.205) between groups using a multivariate repeated measure analysis of variance (ANOVA) with differences between treatment groups assessed over time.
Figure 1.
Average lymphocyte counts relative to day 0 after intranasal challenge with 40 000 cell culture infectious doses of type 2 bovine viral diarrhea virus (BVDV-2) (strain 1373) in calves treated with the antiviral compound DB772 (group A) and untreated (group B) calves.
On day 14, 2 untreated calves (F and H) had platelet counts of less than 100 000/μL (RR: 265 000 to 886 000/μL). Clinically this manifested as ecchymotic hemorrhages on the distal limbs of calf F. The platelet count returned to normal by day 21 in calf F, but remained below the reference range in calf H throughout the study period. Neither thrombocytopenia nor clinical signs of bleeding diatheses were noted in any of the other calves.
Average baseline rectal temperatures obtained after acclimation to study housing and before initiation of treatment were 38.8°C and 39.1°C for treated and untreated calves, respectively (Figure 2). A biphasic fever pattern in untreated calves was evidenced by average rectal temperatures of at least 39.3°C on days 5 and 6 and again on days 9 through 14. Average rectal temperatures were near baseline on days 7 and 8 for the same calves. Three of 4 untreated calves (E, G, and H) exhibited fever of at least 39.8°C on Day 5. Similarly, rectal temperatures of 3 calves (F, G, and H) were consistently 39.6°C to 41.2°C for a 4 d period beginning on day 9. The average daily rectal temperature for treated calves never reached 39.3°C. The average rectal temperature exceeded baseline by more than 0.4°C only on days 16 and 17 at 39.2°C and 39.1°C, respectively. Individually, 1 treated calf exhibited fever over 39.3°C. Calf A had a rectal temperature of 39.7°C on day 16 and 39.6°C on day 17 before returning to baseline. Differences in average rectal temperatures were not statistically significant (P = 0.446) between groups using a multivariate repeated measure ANOVA with differences between treatment groups assessed over time.
Figure 2.
Comparison of the average rectal temperature readings between calves treated with the antiviral compound DB772 (group A) and untreated (group B) calves from day 0 to day 16 after aerosol challenge with type 2 bovine viral diarrhea virus (BVDV). Intranasal challenge with 40 000 cell culture infectious doses of BVDV-2 (1373) occurred on day 0 after the second dose of the antiviral compound was administered.
Virus and antibody detection
Virus was not detected in serum, buffy coat, or nasal swab samples from any calf on days 0 or 2 (Table I). On day 4, the virus was isolated from all of the untreated calves in either buffy coat (calves F and G), serum (E, F, and H), or nasal swab samples (E and F). On days 6 and 8, all samples collected from the untreated calves were positive on virus isolation. Virus was isolated from the buffy coat of 3 calves (F, G, and H) on day 14, but by day 21 there was no virus isolated from samples collected from the untreated calves. No virus was detected in samples from treated calves before day 14. Virus was isolated from the serum and buffy coat of calf A on day 14 only. Calf D had positive virus isolation results on buffy coat on days 21 and 28 and on nasal swab sample on day 28. No virus was detected in samples collected at, or any time prior to, the euthanasia (day 10) or death (day 13) of calves C and B, respectively.
Table I.
Results of virus isolation of BVDV-2 from buffy coat white blood (cell) count (WBC), serum, or nasal swab media (nasal) samples from calves inoculated with 40 000 cell culture infectious doses (CCID50) of BVDV-2 (strain) 1373 by aerosol inoculation on day 0. DB772 was administered to the treatment group intravenously every 8 h for a total of 11 treatments beginning 9 h prior to challenge. Untreated calves were administered an equivalent volume of diluent on the same dosing schedule. One treated calf was euthanized on day 10 and a second calf died on day 13; thus, days 14, 21, and 28 involved only 2 animals in the treated group
| Treated group (positive animals/total animals) | Untreated group (positive animals/total animals) | |||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| WBC | Serum | Nasal | WBC | Serum | Nasal | |
| Day 0 | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) |
| Day 2 | 0/4 (0%) | 0/4 (0%) | N/A | 0/4 (0%) | 0/4 (0%) | N/A |
| Day 4 | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) | 2/4 (50%) | 3/4 (75%) | 2/4 (50%) |
| Day 6 | 0/4 (0%) | 0/4 (0%) | N/A | 4/4 (100%) | 4/4 (100%) | N/A |
| Day 8 | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) | 4/4 (100%) | 4/4 (100%) | 4/4 (100%) |
| Day 14 | 1/2 (50%) | 1/2 (50%) | 0/2 (0%) | 3/4 (75%) | 0/4 (0%) | 0/4 (0%) |
| Day 21 | 1/2 (50%) | 0/2 (0%) | 0/2 (0%) | 0/4 (0%) | 0/4 (0%) | 0/4 (0%) |
| Day 28 | 1/2 (50%) | 0/2 (0%) | 1/2 (50%) | 1/4 (25%) | 0/4 (0%) | 0/4 (0%) |
WBC — white blood cell.
N/A — not available.
All calves were seronegative to BVDV-2 (≤ 1:4) on day 0 (Figure 3). By day 14, all 4 untreated calves developed antibody titers > 1:256 to BVDV-2. On day 21, the titers in untreated calves rose to at least 1:2048 and remained high through the end of the study. Serum antibodies to BVDV-2 were not detected in the treated calves on days 14, 21, or prior to euthanasia or death (calves B and C). Calf A had an antibody titer of 1:256 on day 28, but calf D remained seronegative throughout the study period.
Figure 3.
Serum neutralization titers to type 2 bovine viral diarrhea virus (BVDV-2) in miniature calves after aerosol BVDV challenge. Intranasal challenge with 40 000 cell culture infectious doses of BVDV-2 (1373) occurred on day 0 after the second dose of the antiviral compound was administered. Calves treated with DB772 are denoted by solid lines and untreated calves in are denoted by dashed lines.
Discussion
In this preliminary study, 11 intravenous treatments with DB772 at a dose of 12 mg/kg BW effectively prevented BVDV-associated viremia until long after cessation of therapy in miniature Brahman-cross calves following nasal inoculation with 40 000 CCID50 of BVDV-2 (1373) and continued exposure to infected, untreated calves. Virus was consistently isolated from each untreated calf on days 4, 6, and 8 and from 3 of 4 calves on day 14, consistent with similar previous experimental inoculation studies (19). Virus was not isolated from treated calves until day 14 (calf A) or day 21 (calf D), indicating treatment with DB772 delayed infection with, or viral replication of, BVDV-2 (1373). Though 2 of the treated calves were removed from the study prior to day 14, no virus was isolated from either calf preceding removal from the study or from post-mortem samples. The results suggest DB772 effectively inhibits BVDV infection or initial replication when present in serum at tested concentrations. However, as serum concentrations decline, the calves were once more susceptible to BVDV infection or continuation of initial replication.
In this study, calves were challenged with BVDV after the second administration of DB772 or diluent. Therefore, the serum concentration of DB772 in treated calves was projected to reach or exceed levels sufficient to inhibit BVDV replication in vitro at the time of challenge. Administration of DB772 continued for 72 h after challenge to maintain adequate serum concentrations of the antiviral compound. In a previous study using similar doses of DB772, the serum half-life of DB772 following intravenous injection was reported to be 16.2 h (12). Thus, serum concentrations of DB772 were expected to drop quickly after the final administered dose on day 3.
In the current study protocol, calves were maintained in the same room as an infected untreated calf through the end of the study on day 28. Though the calves were housed in individual crates within the room, the potential for nose-to-nose contact existed when each calf was released from its individual crate into the common room twice a day to allow for cleaning of the individual crates. Virus could still be isolated from 3 of the 4 untreated calves on day 14 of the study, at which time serum DB772 concentrations of their cohorts were projected to be below in vitro protective levels (10). Thus, viral challenge in this study was not limited to a single intranasal inoculation on day 0, but was on-going due to continued viral shedding by the untreated cohorts. We hypothesize that the treated calves were protected from BVDV infection when first exposed by aerosol inoculation, but were left vulnerable to cross-infection from their untreated cohorts in the same room as serum DB772 concentrations waned following the cessation of treatment. A second possibility is that DB772 administration prevented acute viremia and masked clinical signs of disease until therapy ceased and concentrations of the antiviral compound decreased to a degree sufficient to allow progression of the infection.
The first hypothesis is supported by the time of development of serum neutralizing antibodies in the untreated calves compared with their treated cohorts. All untreated calves had serum titers ≥ 1:256 by day 14 and ≥ 1:2048 by day 21, while the 2 remaining treated calves remained seronegative. Serum neutralizing antibodies were not detected in calf A until day 28 (1:256) and calf D remained seronegative throughout the study period. Multiple authors have reported that seroconversion to BVDV occurs within 10 to 21 d after initial viral exposure (20,21). Thus, the delay in the development of serum neutralizing antibodies in treated calves compared to untreated controls suggests that treated calves were not infected by the initial viral challenge but later in the study period after serum concentrations of DB772 had declined below effective levels. Viremia and seroconversion that occurred in treated calves late in the study period suggests treatment with DB772 does not provide long-lasting protection from infection. Though challenged on day 0, treated calves failed to produce adequate amounts of serum neutralizing antibodies to prevent infection from subsequent challenge with BVDV. Consequently, animals treated with DB772 remain susceptible to BVDV after serum antiviral concentrations wane.
An unexpected and concerning result of this study was the development of acute renal toxicity associated with administration of DB772. Renal toxicity was evident in all 4 treated calves and resulted in the death or euthanasia of 2 of the 4 treated calves. This is the first documented report of negative side effects associated with DB772 administration. In previous studies involving healthy calves or calves persistently infected with BVDV, no signs of toxicity were evident clinically or upon evaluation of hematologic parameters (12). Consequently, renal disease associated with DB772 treatment was not foreseen but represents a significant concern that warrants further investigation into the safety of the compound. Earlier safety studies should be repeated with more calves using lower dosages of DB772 to accurately characterize the risk of toxicity to the compound.
An important difference in this study compared with previous in vivo studies was the age and husbandry of the calves treated with DB772 (12). Calves treated previously were 10 to 17 d old and had not yet been weaned. Those calves were allowed to nurse freely from their dams or bottle fed twice daily with a commercial milk replacer. In this study, the calves were 5 to 6 mo old and weaned immediately prior to the housing adjustment period. Though no changes in daily hydration clinical scores or total protein concentrations were appreciated in treated calves, the possibility exists that the calves in the present study were not as well hydrated as previously studied calves, leaving them susceptible to renal insult.
Another difference in this study that may have contributed to the signs of acute renal toxicity was the amount of DB772 administered. In earlier studies, calves were treated with 1.6 mg/kg BW once, 9.5 mg/kg BW every 8 h for 6 d or 12 mg/kg BW every 8 h for 6 d (12). In this study, calves were administered 12 mg/kg BW every 8 h for a total of 11 treatments, a dose equivalent to the highest dose reported previously but for a shorter duration. However, because the calves used in this study were heavier than calves in previous studies, the total cumulative dose of DB772 administered to calves was higher by 2- to 3-fold in some calves herein. Interestingly, the 2 calves most severely affected in this study (calves B and C) were the 2 lightest calves in the treatment group and so received the lowest cumulative doses of DB772.
An important limitation to this study was the small sample size in both the treated and untreated groups. This study was designed as a pilot study to evaluate the antiviral activity of DB772 when administered to healthy calves subsequently challenged intranasally with BVDV. Because of the small sample size, no statistically significant differences were detected in biochemical parameters or rectal temperatures between the treated and untreated groups. To achieve the statistical power necessary to detect the expected differences as statistically significant, many more calves would need to be enrolled in this research. For instance, to achieve a statistical power of 0.80 at α = 0.05 for detecting a 25% difference in a biochemical parameter, which exhibits a standard deviation of 25% of the mean, 17 calves would be needed in each experimental group.
Though clinical signs of BVDV infection were seen in the untreated calves, the severe disease and high mortality rates observed in previous experiments using BVDV-2 (1373)(14,15) were not observed in this study. Previous studies have used both older and younger animals (14,16). The virus titer of the challenge inoculum in our study was 1 to 2 log scores lower than in previously reported studies (14,15,19) and may have accounted for the decreased morbidity and mortality seen in this study. In our study, 2 of the 4 untreated calves developed watery diarrhea and 2 of the 4 also exhibited thrombocytopenia 14 d after experimental inoculation, which manifested as ecchymotic hemorrhages on the distal limb of one calf (calf F). In previous studies using BVDV-2 (1373), the disease was characterized by thrombocytopenia and bloody diarrhea, which was not evident herein. The decrease in severity of clinical signs was thought to be as a result of the lower inoculum used during challenge.
Elevated rectal temperatures were detected in untreated calves on days 5 and 6 and again on days 9 through 14 with the maximum fever being reached on days 10 to 12. Biphasic fever has been previously documented with BVDV infection (20,21) with the maximal rectal temperatures in those studies being reached on day 6 or days 8 and 9, respectively. Fever was not seen in 3 of the calves receiving the antiviral compound; however, calf A spiked a fever on days 16 and 17. Likewise, untreated calves exhibited severe lymphopenia beginning on day 4, similar to what has been described in previous studies also using BVDV-2 (1373)(15,16). While the average lymphocyte count in untreated calves declined 48% by day 4, the average lymphocyte count in treated calves only declined 24% by day 8. These results suggest intravenous treatment with DB772 at 12 mg/kg BW was sufficient to prevent infection or viral replication after intranasal inoculation of BVDV.
Previous in vitro studies indicate the 90% viral inhibitory concentration of DB772 against BVDV-1 is 0.02 μM (9). Similar antiviral efficacy against BVDV-2 is seen with DB772 and closely related compounds (11). The dose used in this study was designed to achieve a 4 μM concentration in serum; this dose has been proven to eliminate BVDV from cells in vitro(10). Serum DB772 concentrations consistently exceeded 3 μM after 24 h in nursing calves on the same dosing regimen (12). While this suggests a lower concentration of DB772 may still be effective at preventing viral replication while minimizing the signs of renal toxicity evident in this study, this remains to be proven in additional studies.
Identifying and eliminating animals persistently infected with BVDV is the cornerstone of BVDV control in North America and much of the world (1). However, an antiviral agent such as, or similar to, DB772 that could prevent BVDV infection as well as maintain the seronegative status of animals exposed to the virus, would be of great benefit in regions free of BVDV where serology is an important component of disease surveillance (22). Maintaining seronegativity in treated animals in North America and other regions where vaccination is widespread may be less crucial because serology is less commonly used as a diagnostic test of BVDV exposure. Further studies must examine the effect of DB772 in seropositive animals. Additionally, the availability of an easily administered specific antiviral compound for use during an outbreak that would maintain the integrity of the diagnostic infrastructure would be invaluable in BVDV-free regions. In this study, DB772 was administered intravenously 3 times a day, which is impractical for commercial or large-scale use. If DB772 or a related compound is to be developed as a prophylactic compound to be used in the face of BVDV outbreaks, a formulation that is easy to administer and demonstrates long-term efficacy must be developed.
In summary, BVDV viremia and clinical signs were effectively prevented in miniature calves during treatment with 12 mg/kg BW of DB772 intravenously every 8 h. However, DB772 administration in this study was associated with signs of acute renal toxicity that resulted in the death or euthanasia in 2 of the 4 treated animals. Because the calculated serum concentrations of DB772 greatly exceeded the concentrations shown to inhibit BVDV in vitro, a lower dose could potentially be used to achieve the same antiviral effects without the negative side effects. Thus, this research proves the concept that an antiviral agent can be used to prevent acute disease caused by BVDV although further investigation is required to ensure that acute renal toxicity does not result from antiviral treatment.
References
- 1.Walz PH, Grooms DL, Passler T, et al. Control of bovine viral diarrhea virus in ruminants. J Vet Intern Med. 2010;24:476–486. doi: 10.1111/j.1939-1676.2010.0502.x. [DOI] [PubMed] [Google Scholar]
- 2.Houe H. Epidemiology of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract. 1995;11:521–547. doi: 10.1016/s0749-0720(15)30465-5. [DOI] [PubMed] [Google Scholar]
- 3.Scruggs DW, Fleming SA, Maslin WR, Groce AW. Osteopetrosis, anemia, thrombocytopenia, and marrow necrosis in beef calves naturally infected with bovine virus diarrhea virus. J Vet Diagn Invest. 1995;7:555–559. doi: 10.1177/104063879500700426. [DOI] [PubMed] [Google Scholar]
- 4.Baker JC. The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract. 1995;11:425–445. doi: 10.1016/s0749-0720(15)30460-6. [DOI] [PubMed] [Google Scholar]
- 5.Rebhun WC, French TW, Perdrizet JA, Dubovi EJ, Dill SG, Karcher LF. Thrombocytopenia associated with acute bovine virus diarrhea infection in cattle. J Vet Intern Med. 1989;3:42–46. doi: 10.1111/j.1939-1676.1989.tb00327.x. [DOI] [PubMed] [Google Scholar]
- 6.Kumar A, Riddell KP, Spychala J, et al. Synthesis of dicationic diarylpyridines as nucleic-acid binding agents. Eur J Med Chem. 1995;30:99–106. doi: 10.1016/0223-5234(96)88214-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Vonderfecht SL, Miskuff RL, Wee SB, et al. Protease inhibitors suppress the in vitro and in vivo replication of rotavirus. J Clin Invest. 1988;82:2011–2016. doi: 10.1172/JCI113821. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Dubovi EJ, Geratz JD, Tidwell RR. Inhibition of respiratory syncytial virus by bis(5-amidino-2-benzimidazolyl)methane. Virol. 1988;103:502–509. doi: 10.1016/0042-6822(80)90207-x. [DOI] [PubMed] [Google Scholar]
- 9.Givens MD, Dykstra CC, Brock KV, et al. Detection of inhibition of bovine viral diarrhea virus by aromatic cationic molecules. Antimicrob Agents Chemother. 2003;47:2223–2230. doi: 10.1128/AAC.47.7.2223-2230.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Givens MD, Stringfellow DA, Dykstra CC, et al. Prevention and elimination of bovine viral diarrhea virus infections in fetal fibroblast cells. Antiviral Res. 2004;64:113–118. doi: 10.1016/j.antiviral.2004.07.004. [DOI] [PubMed] [Google Scholar]
- 11.Givens MD, Galik PK, Riddell KP, Dykstra CC, Brock KV, Stringfellow DA. Effects of aromatic cationic molecules on bovine viral diarrhea virus and embryonic development. Theriogenology. 2005;63:1984–1994. doi: 10.1016/j.theriogenology.2004.09.004. [DOI] [PubMed] [Google Scholar]
- 12.Newcomer BW, Marley MS, Walz PH, Boykin DW, Givens MD. Antiviral treatment of calves persistently infected with bovine viral diarrhoea virus. Antivir Chem Chemother. 2012;22:171–179. doi: 10.3851/IMP1903. [DOI] [PubMed] [Google Scholar]
- 13.Carman S, van Dreumel T, Ridpath J, et al. Severe acute bovine viral diarrhea in Ontario, 1993–1995. J Vet Diagn Invest. 1998;10:27–35. doi: 10.1177/104063879801000106. [DOI] [PubMed] [Google Scholar]
- 14.Brock KV, Widel P, Walz P, Walz HL. Onset of protection from experimental infection with type 2 bovine viral diarrhea virus following vaccination with a modified-live vaccine. Vet Ther. 2007;8:88–96. [PubMed] [Google Scholar]
- 15.Liebler-Tenorio EM, Ridpath JE, Neill JD. Distribution of viral antigen and development of lesions after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves. Am J Vet Res. 2002;63:1575–1584. doi: 10.2460/ajvr.2002.63.1575. [DOI] [PubMed] [Google Scholar]
- 16.Stoffregen B, Bolin SR, Ridpath JF, Pohlenz J. Morphologic lesions in type 2 BVDV infections experimentally induced by strain BVDV2-1373 recovered from a field case. Vet Microbiol. 2000;77:157–162. doi: 10.1016/s0378-1135(00)00272-8. [DOI] [PubMed] [Google Scholar]
- 17.Walz PH, Givens MD, Cochran A, Navarre CB. Effect of dexamethasone administration on bulls with a localized testicular infection with bovine viral diarrhea virus. Can J Vet Res. 2008;72:56–62. [PMC free article] [PubMed] [Google Scholar]
- 18.Givens MD, Heath AM, Brock KV, Brodersen BW, Carson RL, Stringfellow DA. Detection of bovine viral diarrhea virus in semen obtained after inoculation of seronegative postpubertal bulls. Am J Vet Res. 2003;64:428–434. doi: 10.2460/ajvr.2003.64.428. [DOI] [PubMed] [Google Scholar]
- 19.Archambault D, Beliveau C, Couture Y, Carman S. Clinical response and immunomodulation following experimental challenge of calves with type 2 noncytopathogenic bovine viral diarrhea virus. Vet Res. 2000;31:215–227. doi: 10.1051/vetres:2000117. [DOI] [PubMed] [Google Scholar]
- 20.Traven M, Alenius S, Fossum C, Larsson B. Primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf. Zentralbl Veterinarmed B. 1991;38:453–462. doi: 10.1111/j.1439-0450.1991.tb00895.x. [DOI] [PubMed] [Google Scholar]
- 21.Polak MP, Zmudzinski JF. Experimental inoculation of calves with laboratory strains of bovine viral diarrhea virus. Comp Immunol Microbiol Infect Dis. 2000;23:141–151. doi: 10.1016/s0147-9571(99)00060-0. [DOI] [PubMed] [Google Scholar]
- 22.Sandvik T. Progress of control and prevention programs for bovine viral diarrhea virus in Europe. Vet Clin North Am Food Anim Pract. 2004;20:151–169. doi: 10.1016/j.cvfa.2003.12.004. [DOI] [PubMed] [Google Scholar]