M.D. Givens

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.

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.

Infectious agents in bovine embryo production: hazards and solutions.

Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed.

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.

QUANTITY AND INFECTIVITY OF EMBRYO-ASSOCIATED BOVINE VIRAL DIARRHEA VIRUS AND ANTIVIRAL INFLUENCE OF A BLASTOCYST IMPEDE IN VITRO INFECTION OF UTERINE TUBAL CELLS

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7d post-fertilization were exposed (for 2h) to 2 x 10(5-7) cell culture infective dose (CCID(50))/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was <or=6.62+/-1.57 copies/5 microL; 90% of the contaminated embryos had <or=4.64+/-1.57 viral copies/5 microL of embryo-associated virus, using tolerance intervals (P<0.05). The SEM was 0.33 and the mean of averages was 1.12/5 microL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was <or=3.44+/-0.89 copies/5 microL; 90% of the contaminated embryos had <or=2.40+/-0.89 viral copies/5 microL of embryo-associated virus using tolerance intervals (P<0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 microL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.

Efficacy of a recombinant trypsin product against bovine herpesvirus 1 associated with in vivo- and in vitro-derived bovine embryos

Although porcine-origin trypsin will effectively remove bovine herpesvirus 1 (BHV-1) associated with in vivo-derived embryos, TrypLE, a recombinant trypsin-like protease, has not been evaluated. In Experiment 1, 17 groups of 10 in vivo-derived embryos were exposed to BHV-1, treated with TrypLE Express or TrypLE Select (10x concentration) for varying intervals, and assayed as 2 groups of 5 embryos. TrypLE Select treatment for 5 and 10 min (two and seven groups of five embryos, respectively) effectively inactivated BHV-1. In Experiment 2, 22 groups of 10 IVF embryos were treated and assayed. Treatment with TrypLE Select for 7 and 10 min (six groups of five embryos each) and with TrypLE Select diluted 1:2 for 10 min (seven groups of five embryos) was also effective. In Experiment 3, 17 groups of 10 IVF embryos were further evaluated with TrypLE Select undiluted and diluted 1:2 for 10 min. Treatment with the diluted product was effective (18 groups of five embryos), whereas the undiluted product was not completely effective (virus isolated from 2 of 16 groups). In Experiment 4, IVF embryos were treated as described in Experiment 3 and then cultured individually or as groups of five on uterine tubal cells (UTCs) for 48 h; 60% of UTC samples associated with groups of embryos and 35% of UTC associated with individual embryo samples were positive for BHV-1. Therefore, although TrypLE Select appeared to have promise for the treatment of in vivo-derived embryos, it cannot be recommended for treatment of in vitro-derived embryos.

Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo―derived bovine embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo-derived bovine embryos (n=10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID(50))/mL. Additionally, control heifers received 1.5 x 10(6) CCID(50) BVDV/.5 mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.