238 EFFICACY OF RECOMBINANT TRYPSIN AGAINST BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VITRO-DERIVED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 234 ◽  
Author(s):  
M. S. D. Marley ◽  
M. D. Givens ◽  
P. K. Galik ◽  
K. P. Riddell ◽  
D. A. Stringfellow
Keyword(s):  
Virus Isolation ◽  
Bovine Herpesvirus ◽  
Positive Control ◽  
Negative Control ◽  
Animal Origin ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1 ◽  
Porcine Origin

TrypLETM (Invitrogen, Carlsbad, CA, USA) is a recombinant, fungal, trypsin-like protease that is used as a substitute for porcine-origin trypsin in cell culture procedures. It is stable at room temperature and does not present the same risk of contamination as animal-origin trypsin. Previously, TrypLE SelectTM (10X) was shown to remove bovine herpesvirus-1 (BHV-1) from Day 7 in vivo-derived embryos (Marley et al. 2006 Reprod. Fert. Dev. 18, 213–214). The objective of this study was to determine if the same treatment would effectively remove BHV-1 from Day 7 zona pellucida-intact, in vitro-derived porcine embryos. Day 7 in vitro-derived morulae and blastocysts and non-fertile or degenerate embryos (NFD) were washed according to the International Embryo Transfer Society protocol. One group of 10 NFD was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106–108 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 10 NFD was washed and served as the positive control. The remaining developed embryos were divided into groups of 10 and washed and treated as described in Table 1. Following treatment, the embryos were sonicated in groups of 5 and assayed by virus isolation. The negative control embryos, as well as the embryos treated with porcine-origin trypsin, TrypLE Select (10X) for 7 min, and TrypLE Select (10X) diluted 1 : 2 for 10 min, were negative for virus. The positive control embryos in addition to the other treatments were positive on virus isolation (Table 1). Although, TrypLE Select (10X) does have some antiviral effect when used for 10 min, it was not completely effective, as shown by the positive virus isolation results of one group of 10 embryos. The groups treated with TrypLE Select (10X) diluted 1 : 2 for 10 min were negative for virus; however, if a larger sample size had been tested, positive groups might have occurred. Though using a recombinant trypsin product would be beneficial over using an animal-origin product, it is not known if TrypLE Select (10X) would render a single IVF embryo free of infectious virus. Further research would also need to be performed to assess the viability of embryos following treatment with TrypLE Select (10X). In addition, other recombinant trypsin products need to be evaluated to determine their efficacy against BHV-1 associated with IVF embryos. Table 1.Effect of recombinant trypsin-like proteases on BHV-1 virus in porcine embryos

212 TRYPLE SELECT (10X) EFFECTIVELY REMOVES BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VIVO-DERIVED EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 213 ◽  
Author(s):  
M. Marley ◽  
C. Looney ◽  
M. Givens ◽  
P. Galik ◽  
K. Riddell ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Virus Isolation ◽  
Bovine Herpesvirus ◽  
Positive Control ◽  
Negative Control ◽  
Animal Origin ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1 ◽  
Porcine Origin

Porcine-origin trypsin will effectively remove bovine herpesvirus-1 (BHV-1) from in vivo-derived embryos. It is not known if TrypLE (Invitrogen, Carlsbad, CA, USA) could be used to remove BHV-1, but this recombinant porcine sequence trypsin-like protease would be an attractive alternative because it is highly stable at room temperature and does not pose the same threat for contamination as animal-origin trypsin. Thus, the objective of this study was to determine if TrypLE Express (1X) for 1.5 min of exposure or TrypLE Select (10X) for 10 min of exposure would be effective at removing BHV-1 from Day 7 zona pellucida-intact, in vivo-derived embryos after they had been exposed to the virus. Day 7 bovine in vivo-derived morulae and blastocysts and non-fertile degenerate (NFD) embryos were collected and shipped overnight to our facility. Upon arrival, the zona pellucida intact embryos were washed according to the International Embryo Transfer Society protocol. Developed embryos were washed separately from NFD embryos. One group of 5 or 10 NFD or developed embryos was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 5 or 10 NFD or developed embryos was washed and served as the positive control. One group of 10 developed embryos was washed and treated with porcine origin trypsin. The remaining developed embryos were divided into groups of 5 or 10 and washed and treated with TrypLE Express for 1.5 min or TrypLE Select (10X) for 10 min. Following treatment, the embryos were sonicated in groups of 5 or 10 and assayed by virus isolation. The negative control embryos, porcine origin trypsin treated embryos, and TrypLE Select treated embryos were negative for virus. The positive control embryos and the TrypLE Express treated embryos were positive on virus isolation (Table 1). When it was determined that TrypLE Express was not effective at 1.5 min, TrypLE Select (10X) was used for 10 min. These preliminary results indicate that use of TrypLE Select (10X) for 10 min is effective for removal of BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. In addition, TrypLE Select has the advantage of being an animal-origin-free product. However, use of TrypLE Express (1X) for 1.5 min was not effective. Because it is not practical to expose embryos to trypsin for 10 min, further research is needed to determine the ideal treatment concentration and time that will effectively remove BHV-1 without harming in vivo-derived bovine embryos. Table 1. Virus isolation results

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

2008 ◽  
Vol 69 (6) ◽  
pp. 746-757 ◽  
Author(s):  
M.S.D. Marley ◽  
M.D. Givens ◽  
P.K. Galik ◽  
K.P. Riddell ◽  
C.R. Looney ◽  
...  
Keyword(s):  
Bovine Herpesvirus ◽  
Bovine Embryos ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1

scholarly journals Rise and Survival of Bovine Herpesvirus 1 Recombinants after Primary Infection and Reactivation from Latency

2003 ◽  
Vol 77 (23) ◽  
pp. 12535-12542 ◽  
Author(s):  
Frédéric Schynts ◽  
François Meurens ◽  
Bruno Detry ◽  
Alain Vanderplasschen ◽  
Etienne Thiry
Keyword(s):  
Bovine Herpesvirus ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein C ◽  
Recombinant Viruses ◽  
Reactivation Treatment ◽  
Frequent Event ◽  
Herpesvirus 1

ABSTRACT Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC−/gE+) or E (gC+/gE−) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC+/gE+ and gC−/gE−) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC+/gE+ and gC−/gE+) predominated without fully outcompeting the presence of the two other detected populations (gC+/gE− and gC−/gE−), and (iii) after reactivation from latency, no gC+/gE− and gC−/gE− progeny viruses were detected, although gC+/gE− mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.

scholarly journals Glycoprotein D of Bovine Herpesvirus 5 (BoHV-5) Confers an Extended Host Range to BoHV-1 but Does Not Contribute to Invasion of the Brain

2010 ◽  
Vol 84 (11) ◽  
pp. 5583-5593 ◽  
Author(s):  
Evgeni Gabev ◽  
Kurt Tobler ◽  
Carlos Abril ◽  
Monika Hilbe ◽  
Claudia Senn ◽  
...  
Keyword(s):  
Host Range ◽  
Bovine Herpesvirus ◽  
Wild Type ◽  
Bovine Herpesvirus 1 ◽  
High Incidence ◽  
Herpesvirus 1 ◽  
Virulent Phenotype ◽  
The Brain

ABSTRACT Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

scholarly journals Pseudorabies Virus Expressing Bovine Herpesvirus 1 Glycoprotein B Exhibits Altered Neurotropism and Increased Neurovirulence

2000 ◽  
Vol 74 (2) ◽  
pp. 817-827 ◽  
Author(s):  
Volker Gerdts ◽  
Jörg Beyer ◽  
Béla Lomniczi ◽  
Thomas C. Mettenleiter
Keyword(s):  
Respiratory Symptoms ◽  
Pseudorabies Virus ◽  
Bovine Herpesvirus ◽  
Glycoprotein B ◽  
Target Cells ◽  
Homologous Gene ◽  
Wild Type ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1

ABSTRACT Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism ofPseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszky's disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754–2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrV's natural host, swine, 4-week-old piglets were intranasally infected with 106 PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.

211 LACTOFERRIN INHIBITS BOVINE HERPESVIRUS-1 IN CELL CULTURE AND ALLOWS NORMAL DEVELOPMENT OF IN VITRO-PRODUCED EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 213 ◽  
Author(s):  
M. Givens ◽  
M. Marley ◽  
P. Galik ◽  
K. Riddell ◽  
D. Stringfellow
Keyword(s):  
Cell Culture ◽  
Cell Count ◽  
Bovine Milk ◽  
Control Sample ◽  
Bovine Herpesvirus ◽  
Blastocyst Development ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1 ◽  
In Vitro Embryo Production

Lactoferrin is an iron-binding glycoprotein found in milk, saliva, tears, and other exocrine secretions. It is known to have in vitro antiviral effects against human, feline, and canine herpesviruses. In addition, lactoferrin is known to be safe in cell culture. Bovine herpesvirus-1 (BHV-1) is a likely contaminant of in vitro embryo production. Further, trypsin treatment is not completely effective in removing the virus from these embryos. We hypothesized that a nontoxic concentration of lactoferrin might prevent replication of BHV-1 within in vitro embryo production systems. Thus, the specific objectives of this research were to determine if lactoferrin from bovine milk would inhibit BHV-1 in cell culture and to determine if in vitro-produced embryos could develop normally when cultured in lactoferrin. Two-fold dilutions of lactoferrin (from 10 to 0.625 mg/mL) were added to Madin Darby bovine kidney cells, followed in 15 min by the addition 104 PFU/mL of BHV-1 (Colorado strain). Samples of cell lysate were taken at Day 2 and virus was quantified by plaque assay. The percent of virus inhibited by the antiviral agent at each concentration was determined by comparison to equivalent samples from temporal control cultures in which no compound was added before or after inoculation (Percentage of virus inhibited = [Quantity of virus in the control sample - Quantity of virus in the compound sample]/Quantity of virus in the control sample � 100). Next, the effect of lactoferrin was determined on in vitro-produced embryos. Cumulus oocyte complexes were received from an abattoir, matured in transit, placed in fertilization drops for 6 h, and then placed in culture drops containing lactoferrin (10, 5, and 2.5 mg/mL). At Day 3.5, embryos > 4 cell stage were placed into fresh culture drops containing lactoferrin. On Day 7.5, blastocyst development was noted and the developed embryos were stained to count viable cells. Blastocyst development rate and nucleated cell count of the treated embryos were compared to those of the controls using Chi square test, and ANOVA and Tukey-Kramer HSD, respectively. Lactoferrin (10 mg/mL) inhibited 2 to 5 logs of virus. At concentrations of 5 and 2.5 mg/mL, 1 to 3 logs of virus were inhibited, and concentrations of 1.25 and 0.625 mg/mL inhibited 0 to 2 logs of virus. Lactoferrin did not affect the nucleated cell count of the treated embryos. In addition, unlike 10 and 5 mg/mL, 2.5 mg/mL of lactoferrin did not affect blastocyst development. These preliminary results indicate that lactoferrin from bovine milk can significantly inhibit BHV-1 in cell culture. Furthermore, supplementation of in vitro culture with 2.5 mg/mL of lactoferrin does not affect blastocyst development or cell count of in vitro-produced embryos.

148 RISK OF TRANSMISSION OF BOVINE HERPESVIRUS (BHV-1) BY INFECTED SEMEN TO RECIPIENTS AND EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 173 ◽  
Author(s):  
A. Bielanski ◽  
A. Lalonde ◽  
J. Algire
Keyword(s):  
Reproductive Tract ◽  
Bovine Herpesvirus ◽  
Corpora Lutea ◽  
Bovine Herpesvirus 1 ◽  
Bull Semen ◽  
Stage Embryo ◽  
Morula Stage ◽  
Herpesvirus 1 ◽  
Isolation Test

Bovine herpesvirus-1 (BHV-1) causes a variety of economically important respiratory and reproductive problems, the latter including vulvovaginitis, endometritis and infertility. For that reason, several countries have eradicated the disease and others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BHV-1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transter (ET), can lead to the transmission of BHV-1 to recipients and offspring. For this experiment, bull semen contaminated in vitro with BHV-1 at 102 TCID50 mL–1 (Colorado strain) and then cryopreserved was used for insemination (2 times at estrus) of BHV-1 seronegative, superovulated heifers (N = 18). Embryos were collected postmortem 7 days post-insemination and were washed according to the IETS recommendations (however without trypsin treatment) or left unwashed. On 4 occasions, washed embryos were transferred to BHV-1 seronegative recipients. The remaining embryos and other samples collected from the reproductive tract were tested for BHV-1 presence using the standard virus isolation test. In total, out of 144 unfertilized oocytes and embryos collected, 9 were ET quality. Most of the embryos were degenerated (N = 79) or unfertilized (N = 56). The 4 heifers, which each received a single morula-stage embryo, maintained seronegative status, but did not become pregnant. BHV-1 was detected in 43% (23/53) unwashed and 0% (0/57) of washed embryos, 78% (14/18) of follicular fluid samples, 89% (16/18) of oviductal epithelial cells, 78% (14/18) of endometrium, and 89% (16/18) of corpora lutea tissues. Results herein suggest that BHV-1 can be transmitted by infected semen to embryo donors. The resulting unwashed embryos may remain infectious. However, whether BHV-1 uninfected offspring can be produced by ET of BHV-1 contaminated embryos that are washed according to the IETS guidelines, remains to be determined.

scholarly journals Cleaved CD31 as a target for in vivo molecular imaging of inflammation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jonathan Vigne ◽  
Sylvie Bay ◽  
Rachida Aid-Launais ◽  
Guillaume Pariscoat ◽  
Guillaume Rucher ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Molecular Form ◽  
Positive Control ◽  
Negative Control ◽  
Stability Study ◽  
Biodistribution Studies ◽  
Wide Range ◽  
Significant Difference

AbstractThere is a need for new targets to specifically localize inflammatory foci, usable in a wide range of organs. Here, we hypothesized that the cleaved molecular form of CD31 is a suitable target for molecular imaging of inflammation. We evaluated a bioconjugate of D-P8RI, a synthetic peptide that binds all cells with cleaved CD31, in an experimental rat model of sterile acute inflammation. Male Wistar rats were injected with turpentine oil into the gastrocnemius muscle two days before 99mTc-HYNIC-D-P8RI (or its analogue with L-Proline) SPECT/CT or [18F]FDG PET/MRI. Biodistribution, stability study, histology, imaging and autoradiography of 99mTc-HYNIC-D-P8RI were further performed. Biodistribution studies revealed rapid elimination of 99mTc-HYNIC-D-P8RI through renal excretion with almost no uptake from most organs and excellent in vitro and in vivo stability were observed. SPECT/CT imaging showed a significant higher 99mTc-HYNIC-D-P8RI uptake compared with its analogue with L-Proline (negative control) and no significant difference compared with [18F]FDG (positive control). Moreover, autoradiography and histology revealed a co-localization between 99mTc-HYNIC-D-P8RI uptake and inflammatory cell infiltration. 99mTc-HYNIC-D-P8RI constitutes a new tool for the detection and localization of inflammatory sites. Our work suggests that targeting cleaved CD31 is an attractive strategy for the specific in vivo imaging of inflammatory processes.

scholarly journals Brazilian Red Propolis Is as Effective as Amoxicillin in Controlling Red-Complex of Multispecies Subgingival Mature Biofilm In Vitro

Antibiotics ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 432
Author(s):  
Kadmo Azevedo de Figueiredo ◽  
Helio Doyle Pereira da Silva ◽  
Stela Lima Farias Miranda ◽  
Francisco Jerfeson dos Santos Gonçalves ◽  
Arlene Pereira de Sousa ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Positive Control ◽  
Negative Control ◽  
In Vivo Studies ◽  
Complex Species ◽  
Hybridization Data ◽  
Cell Counts ◽  
Actinomyces Species

This study investigated the effects of Brazilian Red Propolis (BRP) extract on seven-day-old multispecies subgingival biofilms. Mixed biofilm cultures containing 31 species associated with periodontal health or disease were grown for six days on a Calgary device. Then, mature biofilms were treated for 24 h with BRP extract at different concentrations (200–1600 µg/mL), amoxicillin (AMOXI) at 54 µg/mL (positive control) or vehicle (negative control). Biofilm metabolic activity was determined by colorimetry, and bacterial counts/proportions were determined by DNA–DNA hybridization. Data were analyzed by Kruskal–Wallis and Dunn’s tests. Treatment with BRP at 1600, 800 and 400 μg/mL reduced biofilm metabolic activity by 56%, 56% and 57%, respectively, as compared to 65% reduction obtained with AMOXI. Mean total cell counts were significantly reduced in all test groups (~50–55%). Lower proportions of red, green and yellow complex species were observed upon treatment with BRP (400 µg/mL) and AMOXI, but only AMOXI reduced the proportions of Actinomyces species. In conclusion, BRP extract was as effective as AMOXI in killing seven-day-old multispecies biofilm pathogens and did not affect the levels of the host-compatible Actinomyces species. These data suggest that BRP may be an alternative to AMOXI as an adjunct in periodontal therapy. In vivo studies are needed to validate these results.

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