Kenny V. Brock

Persistent bovine viral diarrhoea virus infection in US beef herds

In the summer of 1996, we screened 18,931 calves in 128 beef herds located in five US states for persistent bovine viral diarrhea virus (BVDV) infection. Of these, 76 herds were randomly selected from the client database of collaborating veterinary practices, and 52 herds were suspected by the collaborating veterinarians to have BVDV infection based on history or clinical signs. Serum was obtained from each calf in the cooperating herds prior to 4 months of age and tested for the presence of BVDV by microtiter virus isolation. Information about each of the herds (including management practices, vaccination history, and breeding- and calving-season production measures) were collected by the collaborating veterinarians using standardized questionnaires. A total of 56 BVDV-positive calves in 13 herds were identified on initial screening. Ten (19%) of the BVDV-suspect herds and three (4%) of the randomly selected herds had > or = 1 BVDV-positive calf at initial screening. Multiple BVDV-positive calves were identified in 10 of those 13 herds. Follow-up information was obtained for 54 of the 56 positive calves. Ten out of 54 (18%) died prior to weaning, and 1 (2%) was sold because of unusually poor growth. Thirty-three out of 54 (61%) of the initially positive calves remained BVDV positive at 6 months of age - confirming persistent-infection (PI) status. Dams of 45 of the 56 positive calves were tested, with 3 (7%) identified as positive - indicating most PI calves were products of acute dam infection during gestation. The proportion of cows that were pregnant at the fall 1995 pregnancy examination was 5% lower in herds with PI calves born during the 1996 calving season than in randomly selected herds without PI calves. Most of the calves we identified with persistent BVDV infections survived to weaning, and could provide a constant source of virus to the herd throughout the breeding season and early gestation.

The persistence of bovine viral diarrhea virus

Bovine viral diarrhoea virus (BVDV) has a unique capacity to cause persistent infections of foetuses exposed within the first 150 days of gestation. Preventing foetal BVDV infection will aid in improved control. This unique ability gives BVDV a selective advantage allowing continual mutation and antigenic variation within cattle populations. Therefore, BVDV has become widespread and causes economic losses due to respiratory, reproductive and enteric disease. Vaccination (modified-live or killed) can provide some protection from acute disease and the development of persistently infected foetuses. However, vaccination programmes alone cannot control or eliminate BVDV. In naturally exposed and vaccinated herds, BVDV infections are not self-limiting and may persistent over time. This underscores the ability of the BVDV genome to remain fluid and adapt under selective pressures. Factors influencing persistence of BVDV infections in cattle populations include: non-lytic infections; evasion of host immune responses; foetal infections; acute infections; management practices; contaminated biologics; secondary hosts; defective replicated intermediates; antigenic variation; and replication in privileged anatomical sites.

Quantitation of bovine viral diarrhea virus in embryo transfer flush fluids collected from a persistently infected heifer

Due to the epidemiological significance of persistently infected animals, it is important that possible methods of transmission be investigated. Currently, the recommended prevention and control measures for BVDV are centered around the identification and removal of persistently infected animals and the judicious use of BVDV vaccines. 4,6 Unfortunately, the identification of persistently infected animals has not been emphasized until recently and remains impractical. The purpose of this report is to emphasize the epidemiological significance of animals persistently infected with BVDV, especially in relation to embryo transfer procedures. A 5-month-old Holstein heifer, persistently infected with BVDV, was identified in a dairy herd with a history of BVDV infection by virus isolation from nasal and vaginal swabs and serum. The heifer had no detectable levels of BVDV serum antibodies (in indirect fluorescence assay [IFA]) and was kept in isolation for observation and testing. Estrus first was detected at 14 months of age. Following the third heat cycle the heifer was superovulated with follicle-stimulating hormone (days 11-14) and inseminated with BVDV-negative semen (confirmed negative by virus isolation) during the subsequent estrous. Embryo collection was attempted on day 7 (estrus day 0) by standard whole body uterine flush using 2 liters of Dulbecco’s phosphate-buffered saline containing 3% BVDV-negative fetal bovine serum (confirmed negative by virus isolation). Virus was not isolated from the flush medium prior to the uterine flush. Only 3 unfertile eggs were identified by microscopic examination in the uterine flush fluid (2 liters). The titers of BVDV were quantitated in various samples collected from the persistently infected heifer by virus isolation from 10-fold dilutions of the samples. One milliliter of each dilution was inoculated in replicates of 3 onto BVDVnegative secondary bovine turbinate cells passaged 5-10 times in Dulbecco’s minimum essential medium containing 10% horse serum. The inoculum was removed after 1 hour and

Detection of bovine viral diarrhea virus in the ovaries of cattle acutely infected with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is recognized worldwide as a major cause of economic loss in cattle. Infection with BVDV can result in several clinical outcomes. However, the reproductive consequences may be the most important. Infertility, early embryonic death, abortion, and congenital anomalies have all been reported following acute infection with BVDV. The cause of infertility following acute BVDV infection is not known. BVDV has been isolated from the bovine ovary and has been associated with chronic oophoritis. The purpose of this study was to identify the ovarian cell types infected with BVDV following acute infection. Twelve heifers were acutely infected with noncytopathic BVDV, and ovariectomies were performed between 4 and 60 days postinfection. BVDV was isolated on days 6 and 8 postinfection. Viral antigen was detected in macrophage-like cells and stromal cells in the ovarian cortex and oophoritis was evident from 6 to 60 days postinfection. These findings indicate that acute infection with BVDV may result in changes in ovarian function that could lead to reduced fertility.

Replication and persistence of different strains of bovine viral diarrhea virus in an in vitro embryo production system.

Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.

Identification of a Pegivirus (GB Virus-Like Virus) That Infects Horses

ABSTRACT The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.

Cohabitation of pregnant white-tailed deer and cattle persistently infected with Bovine viral diarrhea virus results in persistently infected fawns.

Economic losses due to infection with Bovine viral diarrhea virus (BVDV) have prompted introduction of organized control programs. These programs primarily focus on the removal of persistently infected (PI) animals, the main source of BVDV transmission. Recently, persistent BVDV infection was demonstrated experimentally in white-tailed deer, the most abundant wild ruminant in North America. Contact of cattle and white-tailed deer may result in interspecific BVDV transmission and birth of persistently infected offspring that could be a threat to control programs. The objective of this study was to assess the potential for interspecific BVDV transmission from persistently infected cattle cohabitated with pregnant white-tailed deer. Seven female and one male white-tailed deer were captured and bred in captivity. At approximately 50 days of gestation, two cattle persistently infected with BVDV 1 were cohabitated with the deer. In a pen of approximately 0.8 ha, both species shared food and water sources for a period of 60 days. Transmission of BVDV as indicated by seroconversion was demonstrated in all exposed adult deer. Of the seven pregnancies, four resulted in offspring that were infected with BVDV. Persistent infection was demonstrated in three singlet fawns by immunohistochemistry and ELISA on skin samples, PCR, and virus isolation procedures. Furthermore, two stillborn fetuses were apparently persistently infected. This is the first report of BVDV transmission from cattle to white-tailed deer using a model of natural challenge. Under appropriate circumstances, BVDV may efficiently cross the species barrier to cause transplacental infection and persistently infected offspring in a wildlife species.

Characterization of nonprimate hepacivirus and construction of a functional molecular clone

Significance The origin of hepatitis C virus (HCV) has long remained a mystery. Unexpectedly, a plethora of HCV-related hepaciviruses was recently discovered in horses, monkeys, rodents, and bats. These discoveries are of particular interest and may aid in understanding HCV evolution, molecular biology, and natural history. Currently, immunocompetent HCV animal models are lacking, impeding vaccine development; novel hepaciviruses and their natural hosts could provide such models. Here, we demonstrate that the closest HCV homolog, nonprimate hepacivirus (NPHV), is a hepatotropic equine virus with many similarities to HCV, including the capacity to establish persistent infection, delayed-onset seroconversion, and liver pathology. We identify the complete NPHV genome and establish a functional clone infectious in horses, a key advance providing a direct link between virus infection and clinical outcome. Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host.

Detection of Inhibition of Bovine Viral Diarrhea Virus by Aromatic Cationic Molecules

ABSTRACT Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle and a problematic contaminant in the laboratory. BVDV is often used as an in vitro model for hepatitis C virus during drug discovery efforts. Aromatic dicationic molecules have exhibited inhibitory activity against several RNA viruses. Thus, the purpose of this research was to develop and apply a method for screening the aromatic cationic compounds for in vitro cytotoxicity and activity against a noncytopathic strain of BVDV. The screening method evaluated the concentration of BVDV in medium and cell lysates after 72 h of cell culture in the presence of either a 25 or 5 μM concentration of the test compound. Five of 93 screened compounds were selected for further determination of inhibitory (90 and 50%) and cytotoxic (50 and 10%) concentration endpoints. The screening method identified compounds that exhibited inhibition of BVDV at nanomolar concentrations while exhibiting no cytotoxicity at 25 μM concentrations. The leading compounds require further investigation to determine their mechanism of action, in vivo activity, and specific activity against hepatitis C virus.

Analytical sensitivity of assays used for detection of bovine viral diarrhea virus in semen samples from the Southeastern United States

Bovine viral diarrhea virus (BVDV) is a significant pathogen that can be shed in the semen of infected bulls. Thus, screening for BVDV in semen of bulls is recommended prior to their entry into an artificial insemination center. No previous research has compared the analytical sensitivity of reverse transcription-nested polymerase chain reaction (RT-nPCR) and virus isolation assays for detection of BVDV in semen from an infected bull. Therefore, the goals of this research were to compare the analytical sensitivity of RT-nPCR and virus isolation assays for BVDV in semen and to apply these assays to determine the prevalence in the Southeastern United States of bulls that lack viremia yet shed BVDV in semen. Semen collected from a bull that was persistently infected with BVDV was serially diluted (1/10) in semen from uninfected bulls and frozen in liquid nitrogen as raw, partially extended or fully extended semen. Subsequently, samples of semen were assayed by virus isolation and RT-nPCR. Viral detection was more sensitive in extended semen samples than in raw semen samples and more sensitive by RT-nPCR than virus isolation. After this evaluation of analytical sensitivity, serum and semen were collected from 558 post-pubertal bulls in our region. These samples were tested for BVDV by virus isolation. Partially extended semen was also assayed for BVDV by RT-nPCR. All samples were negative by all assays for BVDV. The application of analytically sensitive assays reveals a very low prevalence (</=0.54%) of BVDV in semen from bulls in the Southeastern United States.