Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.     

Effect of alpha-MSH on the corticosteroid production of isolated zona glomerulosa and zona fasciculata cells

α-MSH (10^?9 ? 6×10^?7M) potentiates the effect of ACTH (10^?11 ? 5×10^?9M) on adrenocortical steroidogenesis decreassng ED₅₀ of ACTH from 220 to 183 pg/ml on zona fasciculata corticosterone-, and from 739 to 437 pg/ml on zona glomerulosa aldosterone production. α-MSH alone increases aldosterone production of zona glomerulosa cells in doses (10^?9 ? 6×10^?7M) that do not stimulate zona fasciculata corticosterone production. The response of zona glomerulosa aldosterone production to α-MSH can be characterized by a bi-phase dose-response curve.     

Effects of interferon and encephalomyocarditis virus on in vitro development of preimplantation mouse embryos with and without the zona pellucida

Development of preimplantation mouse embryos, with or without the zona pellucida, in the presence of interferon (IFN) and mouse encephalomyocarditis (EMC) virus was studied using the in vitro culture method. The embryos (2- to 8-cell stages) were obtained from superovulated mice and cultured in modified Witten's medium under paraffin oil in 5% CO2 in air at 37 degrees C. Removal of the zona pellucida does not affect the subsequent development of the embryos: 90% of embryos with and 87% of embryos without the zona pellucida reached the morula-early blastocyst stages. Mouse IFN (10(4) units/ml) had no inhibitory effect on the developmental ability of the preimplantation embryos with or without the zona pellucida: 88 and 89% of the embryos in each group, respectively, reached the morula-early blastocyst stages. The preimplantation mouse embryos were sensitive to the embryotoxic effect of EMC virus: at a multiplicity of 20 infection particles per embryo the development of 43% of embryos was inhibited. The zona pellucida had no significant protective effect: Its removal changed only slightly the susceptibility of the preimplantation embryos to this virus. Pretreatment of embryos with IFN did not protect them from the embryotoxic effect of EMC virus. This work indicates that preimplantation mouse embryos appear to be resistant for both the antiviral and antiproliferative activities of IFN.     

Can bluetongue virus (BTV) be transmitted via caprine embryo transfer?

The three objectives of this study were to investigate whether cells of early goat embryos isolated from in vivo fertilized goats interact with bluetongue virus (BTV) in vitro, whether the embryonic zona pellucida (ZP) protects early embryo cells from BTV infection, and whether the 10 wash cycles recommended by the International Embryo Transfer Society (IETS) for bovine embryos effectively decontaminates caprine embryos exposed to Bluetongue Virus (BTV) in vitro. Donor goats and bucks were individually screened and tested negative for the virus by RT-PCR detection of BTV RNA in circulating erythrocytes. ZP-free and ZP-intact 8-16 cell embryos were co-cultured for 36 h in an insert over a Vero cell monolayer infected with BTV. Embryos were washed 10 times in accordance with IETS recommendations for ruminant and porcine embryos, before being transferred to an insert on BTV indicator Vero cells for 6 h, to detect any cytopathic effects (CPE). They were then washed and cultured in B2 Ménézo for 24 h. Non-inoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls.The Vero cell monolayer used as feeder cells for BTV inoculated ZP-free and ZP-intact embryos showed cytopathic effects (CPE). BTV was found by RT-qPCR in the ten washes of exposed ZP-free and ZP-intact embryos. In the acellular medium, the early embryonic cells produced at least 10^2.5 TCID₅₀/ml. BTV RNA was detected in ZP-free and ZP-intact embryos using RT-qPCR.All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with BTV and that infection with this virus is productive. The washing procedure failed to remove BTV, which indicates that BTV could bind to the zona pellucida.     

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection.Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks.This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos.This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10³ TCID₅₀/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.     

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

Resistance of the European catfish (Silurus glanis) to channel catfish virus

European catfish (Silurus glanis) fingerlings (2 to 4 g each) were tested for susceptibility to channel catfish virus (CCV). They had supported CCV replication at 2 days after intraperitoneal injection with 0.1 ml of saline containing 10⁵ TCID₅₀. Homogenized visceral organs (liver, kidney and spleen) contained 10⁴ TCID₅₀/0.1 ml at 2 days post inoculation (PI) but at 4 days the titer decreased to 10¹ TCID₅₀. Bathing European catfish in CCV yielded only one positive sample with à titer of 10^0.83 TCID₅₀ per 0.1 ml of tissue. No clinical signs of CCV developed and no virus related deaths occurred.     

Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos.

In previous experiments, zona pellucida (ZP)-intact in vitro-produced (IVP) embryos incubated for 1 hr with 10(6.3) TCID(50)/ml bovine herpes virus-1 (BHV-1), 10(5.3) TCID(50)/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 10(5.3) TCID(50)/ml noncytopathic (NCP) BVDV showed no signs of virus replication or embryonic degeneration. The aims of the present study were to investigate whether a prolonged presence (24 hr or 8 days) of 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml BVDV in an in vitro embryo production system affected the rate of cleavage and embryonic development of ZP-intact embryos, and to point out eventual causes of adverse effects. When virus was present in each step of an IVP system, significantly lower rates of cleavage and blastocyst formation of virus-exposed embryos were observed, in comparison with control embryos (P < 0.01). When embryos were only exposed to virus during the in vitro fertilization (IVF), the rates of cleavage and blastocyst formation were significantly affected. The introduction of BHV-1 or BVDV during in vitro maturation (IVM) or in vitro culture (IVC) resulted only in significantly lower rates of blastocyst (P < 0.01). In all experiments, virus replication was not detected in the embryonic cells. On the other hand, virus replication was clearly demonstrated in oviductal cells in the co-culture system, resulting in a degeneration of these cells. In an additional experiment, synthetic oviduct fluid (SOF) without somatic cells was used as an alternative culture system. Even when SOF-embryos were exposed to 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml CP, and NCP BVDV, the rates of blastocyst formation of the BHV-1-, CP-, and NCP BVDV-exposed embryos were not different from the unexposed control embryos, 23%, 24%, and 24%, respectively, vs. 27%. Taken together, it can be concluded that the virus-induced adverse effects on embryonic development in conventional co-cultures were due to changes in the embryonic environment caused by infection of oviductal cells.     

Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine embryos to foot-and-mouth disease virus

When 169 zona pellucida-intact bovine embryos were exposed to 10(6) pfu/ml of foot-and-mouth disease virus and then washed, no infectious virus was detected on any of the embryos. FMD viral infectivity was found, however, in association with 14 of 42 hatched (zona pellucida-free) bovine embryos and in a small number of zona pellucida-intact porcine embryos. The porcine embryos were assayed individually and in groups of 8 embryos. Four of the 124 individual embryos and 2 of the 9 groups of embryos carried the infectious virus.     

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 10^2.55TCID₅₀/ml, group B 10^5.25TCID₅₀/ml and group C 10^6.7TCID ₅₀/ml. A fourth group (D) was inoculated with a medium dose (10^5.25TCID₅₀/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.     

Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale)

Melon necrotic leaf spot virus (MNSV) caused a major outbreak of a leaf necrosis disease of hydroponically-grown cucumber plants at Humberside in 1983. The virus had c. 33 nm diam. particles which reacted serologically with MNSV antiserum of Dutch or American origin. Virus particles, which contained a single polypeptide (mol. wt 45 × 10³) and a presumed RNA species (mol. wt 1.5 × 10⁶), had a sedimentation coefficient (s_20.w) of 134 S and a buoyant density in caesium chloride of 1.35 g/cm³. The virus was mechanically transmissible, confined to species of Cucurbitaceae, transmitted by zoospores of Olpidium radicale and retained in the resting spores of the fungus. MNSV is thus both water-borne and soil-borne. O. radicale zoospores were killed in <5 min in suspensions containing 20 μg/ml of the surfactant Agral (alkyl phenol ethylene oxide). The disease did not reappear in 1984 when the cucumber crops were fed with nutrients containing 20μg/ml Agral.     

Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.     

Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1

The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10(7.7) TCID(50)/ml BHV-1 (Cooper strain). Three titers (10(5.7), 10(6.7) and 10(7.7) TCID(50)/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10(1.32) to 10(1.43) TCID(50)/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10(3.08) TCID(50)/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.     

Production of selected baculoviruses in newly established lepidopteran cell lines

Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×10⁶TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×10⁶TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

Pathology and Properties of the Tetravirus Helicoverpa armigera Stunt Virus

A quantitative study of the pathogenicity of Helicoverpa armigera stunt virus (HaSV) (Tetraviridae) isolates toward larvae of several heliothine species was conducted along with studies on the stability of the virus to a variety of chemical, enzymic, and temperature treatments. Surface contamination bioassays of several HaSV isolates against H. armigera produced 50% effective concentration (EC₅₀) estimates ranging between 568 and 9244 virus particles (vp)/mm². Against mid 1st instar larvae of H. armigera, H. punctigera, and Heliothis punctifera, EC₅₀ estimates for one isolate were 1288, 16,137, and 2667 vp/mm², respectively. The virulence of HaSV infection varied markedly with the age at which larvae were exposed to the virus. Presentation of the virus to the first three instars of H. armigera was accompanied by cessation of feeding, growth retardation, and eventual lethality, whereas no adverse effects were observed when later instars were exposed to the virus, even at very high concentrations. Active HaSV was recovered from frass of larvae exposed to the virus as 1st instars. Household bleach (1% v/v; 0.04% w/v available chlorine, 0.004% w/v NaOH), formaldehyde (1% w/v), and temperatures ≥65°C completely inactivated HaSV in suspension. Treatments with ether, proteinase K (1 mg/ml), H. armigera gut contents, and temperatures between 22 and 55°C partially inactivated virus activity. No observable inactivation was observed after treatment with chloroform, chymotrypsin (1 mg/ml), trypsin (1 mg/ml), or RNase A (1 mg/ml). The virus was stable between pH 2.8 and pH 10.0 with around 60% loss of activity observed at pH 11.4. The pattern of pathogenic effects seen in several other insect species challenged by high concentrations of HaSV indicated that the host range of the virus is limited to species within the lepidopteran family Noctuidae. The apparently restricted host range of HaSV along with a number of other features indicate that this virus has considerable potential for the development of novel control agents for use against heliothine pests.     

Effects of cell cycle specific exposure to 3H-thymidine or 3H-arginine on development and cell proliferation of mouse embryos

One-cell mouse embryos were exposed to either ³H-thymidine (100 or 200 kBq/ml) or ³H-arginine (2.5 to 50 kBq/ml) for 2 h either in G₁, S or G₂ phase. ³H-Arginine affected embryonic development and cell proliferation in an activity-dependent way irrespective of the cell cycle stage exposed, whereas ³H-thymidine was effective only at higher activities and only after exposure during S phase. Received: 12 July 1996 / Accepted in revised form: 30 September 1996     

Proliferation and Teratogenicity of Aino Virus in Chick Embryos

Aino virus (AIV; JaNAr 28 strain) 10³ TCID₅₀/0.2 ml was inoculated in the yolk sac of 8-day-old chick embryos. Recovery and titration of the virus from various organs including the central nervous system (CNS) and skeletal muscle were performed at 2, 4, 7, 10 and 13 days after inoculation (PI). AIV was systemically disseminated and proliferated even 2 days PI. The titers of the recovered virus from the CNS and from skeletal muscle was the highest at 4 days PI and declined with time, whereas hydranencephaly, arthrogryposis and cerebellar hypoplasia developed at 7 days PI and gradually progressed until 13 days PI.     

Embryo transfer as a means of controlling the transmission of viral infections. IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

When zona pellucida-intact porcine embryos were exposed to 10(7) plaque-forming units (pfu)/ml of swine vesicular disease virus (SVDV) and then washed, infectious virus could be isolated from all of the embryos. Culturing the embryos for 24 or 48 h or treating the embryos with pronase, trypsin, or antiserum after virus exposure and washing reduced the number of embryos carrying virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of disinfecting every embryo.     

Effect of the bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: A preliminary study

Five crossbred-Holstein cows, approximately three to seven years of age, were superovulated using pregnant mare's serum gonadotropin (PMSG) and prostaglandin F_2α (Prostin F_2α). At the induced estrus, each cow was artificially inseminated with frozen semen. Seven days after insemination, the lumen of the right uterine horn of each cow was inoculated with BVD virus in Eagle's minimum essential tissue culture medium, and the lumen of the left horn was infused with tissue culture medium only. Three days later, each cow was subjected to midventral laparotomy under general anesthesia and embryos were collected. A total of 22 embryos were recovered; 12 were from infected uterine horns and ten were from non-infected uterine horns. All embryos from the non-infected uterine horns were in the late blastocyst stage without the zona pellucida. Of the embryos collected from the infected uterine horns, eight of 12 (66.6%) still possessed zona pellucida and appeared in a degenerative state. The remaining four embryos were morphologically similar to those from the non-infected uterine horns. Electron microscopic examination of the degenerated embryos from the infected uterine horns demonstrated the presence of a structure which morphologically resembled the BVD virus. The results of this preliminary study indicate that the BVD virus within the uterine horns may interfere with normal development of preimplantation bovine embryos. Therefore, it is proposed that the BVD virus could adversely affect early stages of gestation in the cow, resulting in infertility.