Passage In Cell Culture Research Articles

Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors

Cells of the female reproductive tract are subject to hormonal control via sex steroid genomic receptors expressed at nuclear level. We previously showed that interstitial Cajal-like cells (ICLC) of human myometrium expressed estrogen and progesterone receptors (ER/PR). Our aim, based on these results, was to see if ER and/or PR could be found also in tubal ICLC. Indeed, we present here immunohistochemical evidence that ICLC of human Fallopian tube (isthmic region) have such receptors. Stromal ICLC, as well as ICLC among smooth muscle layers, were identified in tissue sections by their morphological features (e.g. several very long, moniliform, prolongations of cell body) as well as by c-kit positivity, vital staining with methylene blue or silver impregnation. Additional evidence was provided by sequential staining for c-kit and for PR on the same cell, by 'sandwich method'. In vitro, the 4th passage cell cultures from Fallopian tube muscularis exhibiting ICLC morphology showed the presence of ER-alpha and/or PR-A by immunofluorescence. In conclusion, our data suggest that ICLC could function as steroid sensors, and might be implicated in Fallopian tube motility (via gap junctions or juxta- and/or paracrine mechanisms).

Evaluation of a protocol for rapid diagnosis of enterovirus associated with acute flaccid paralysis cases

Background The virological surveillance of acute flaccid paralysis (AFP) is a critical component of the initiative of the World Health Organization (WHO) to eradicate poliomyelitis worldwide. Furthermore rapid methods are needed either to detect or rule out the presence of polioviruses during the late stages of eradication, especially in polio-free areas. Objectives The aim of this study was to evaluate a fast protocol combining one passage (5 days) in cell culture followed by RT-PCR and molecular typing in order to detect and type poliovirus (PV) and other enteroviruses associated with AFP cases. Study design A total of 216 fecal suspensions from AFP suspected cases were tested by using this approach and compared with the WHO gold standard. Results Using the WHO protocol enterovirus was detected in 12 out of the 216 AFP samples (5.55%) while with the proposed protocol enterovirus was detected in 15 out of the 216 AFP samples (6.94%). The additional positive samples detected by the proposed method were classified as non-polio enteroviruses (NPEV). Conclusions The proposed protocol showed higher sensitivity than the WHO gold standard, reducing the entire process of identification and typing of the isolates from the typically 14–21 days to only ∼6–8 days.

Paraparesis in a Polar Bear (Ursus maritimus) Associated with West Nile Virus Infection

A polar bear (Ursus maritimus) housed at the Toronto Zoo presented with acute-onset, nonambulatory paraparesis. Physical examination 24 hr after onset was otherwise unremarkable, spinal radiographs looked normal, and blood tests indicated mild dehydration. With continued deterioration in its general condition, euthanasia was elected a day later. Necropsy did not reveal a cause for the major presenting clinical signs. Serum collected at the time of initial examination was positive for West Nile virus (WNV) antibodies in a serum neutralization assay and at the time of euthanasia was positive in both a competitive enzyme-linked immunosorbent assay and in a plaque reduction neutralization assay. The major microscopic finding was a mild-to-moderate nonsuppurative meningoencephalomyelitis. WNV was not detected by immunohistochemistry in brain or spinal cord or by real-time reverse transcription-polymerase chain reaction (RT-PCR) and cell culture of brain and kidney, but it was isolated and identified by RT-PCR in second passage cell culture of spleen. Retrospective immunohistochemistry on spleen revealed rare antigen-positive cells, probably macrophages. Prevention of exposure to potentially WNV-infected mosquitoes or vaccination of captive bears against WNV should be considered.

Design and evaluation of multi-gene, multi-clade HIV-1 MVA vaccines

Recombinant modified vaccinia virus Ankara (rMVA) expressing HIV-1 genes are promising vaccine candidates. Toward the goal of conducting clinical trials with one or a cocktail of recombinant viruses, four rMVAs expressing env and gag-pol genes from primary HIV-1 isolates representing predominant subtypes from Kenya, Tanzania, Uganda, and Thailand (A, C, D, and CRF01_AE, respectively) were constructed. Efficient expression, processing, and function of Env and Gag were demonstrated. All inserted genes were shown to be genetically stable after repeated passage in cell culture. Strong HIV-specific cellular and humoral immune responses were elicited in mice immunized with each individual vaccine candidate. The MVA/CMDR vaccine candidate expressing CRF01_AE genes has elicited HIV-specific T-cell responses in two independent Phase I clinical trials. Further testing of the other rMVA is warranted.

Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.

Angiogenic Potential of Human Dental Pulp Stromal (STEM) Cells

Dental pulp is a heterogeneous microenviroment where unipotent progenitor and pluripotent mesenchymal stem cells cohabit. In this study we investigated whether human dental pulp stromal (stem) cells (DP-SCs) committed to the angiogenic fate. DP-SCs showed the specific mesenchymal immunophenotypical profile positive for CD29, CD44, CD73, CD105, CD166 and negative for CD14, CD34, CD45, in accordance with that reported for bone marrow-derived SCs. The Oct-4 expression in DP-SCs, evaluated through RT-PCR analysis, increased in relation with the number of the passages in cell culture and decreased after angiogenic induction. In agreement with their multipotency, DP-SCs differentiated toward osteogenic and adipogenic commitments. In angiogenic experiments, differentiation of DP-SCs, through vascular endothelial growth factor (VEGF) induction, was evaluated by in vitro matrigel assay and by cytometric analysis. Accordingly, endothelial-specific markers like Flt-1 and KDR were basally expressed and they increased after exposure to VEGF together with the occurrence of ICAM-1 and von Willebrand factor positive cells. In addition, VEGF-induced DP-SCs maintained endothelial cell-like features when cultured in a 3-D fibrin mesh, displaying focal organization into capillary-like structures. The DP-SC angiogenic potential may prove a remarkable tool for novel approaches to developing tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Identification of amino acid residues of Ljungan virus VP0 and VP1 associated with cytolytic replication in cultured cells

Ljungan virus is a picornavirus isolated from Swedish and North American rodents. Replication of Ljungan virus in cultured cells normally induces a weak and delayed cytopathic effect compared to that of many other picornaviruses. However, efficiently replicating Ljungan virus variants may evolve during serial passages in cell culture. In this study, we evaluate the significance of three substitutions in capsid protein VP0 and VP1 of a cell-culture-adapted variant of the Swedish Ljungan virus 145SL strain. In contrast to the parental strain, this 145SLG variant grows to high titers in green monkey kidney cells and induces a distinct cytopathic effect. Reverse genetic analyses demonstrated that each one of the individual capsid substitutions contributes to lytic replication in cell culture, but also that expression of all three substitutions results in a 100- to 500-fold increase in viral titers compared to viruses encoding single capsid substitutions. In addition, as indicated by detection of activated caspase-3 and DNA fragmentation, there seems to be an association between increased replication efficiency of lytic Ljungan virus variants and induction of an apoptotic response in infected green monkey kidney cells.

Cytogenetic abnormalities in mesenchymal stem cells in chronic lymphocytic leukemia (CLL) patients and normal subjects

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]

Odontogenic Potential of Mesenchymal Cells from Hair Follicle Dermal Papilla

Dental pulp stem cells from teeth can be used for tooth regeneration. Although nondental stem cells derived from bone marrow can differentiate into odontoblast-like cells when recombined with embryonic oral epithelium, these cells can lose their ability to differentiate after an extended number of cell culture passages. There has been limited research to identify stem cells from other tissue sources to regenerate teeth. As another candidate source for mesenchymal stem cells, hair follicle has obtained much more attention recently because of its easy accessibility. In this study, cultured vibrissae follicle dermal papilla mesenchymal cells (FDPMCs) from adult C57BL/6 GFP mice can differentiate into adipocytes and osteoblasts in vitro. Moreover, in the inductive microenvironment generated by apical bud and dental mesenchyme from 7-day-old C57 mice, FDPMCs in vitro demonstrated odontogenic potential, as indicated by the morphological transformation, cell-cycle change and expression of tooth-specific markers. Under the same microenvironment, FDPMCs were incubated in vivo for 3 weeks. Coexpression of GFP and DSP proteins in the odontoblast layer was detected in the recovered implants, suggesting that GFP(+) FDPMCs can function as odontoblasts in vivo. Together, our data indicate for the first time that whisker FDPMCs from adult mice can differentiate to odontoblast-like cells.

Properties and use of novel replication-competent vectors based on Semliki Forest virus

BackgroundSemliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important.ResultsWe analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture.ConclusionThe properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

In vitro and in vivo genetic stability studies of a human adenovirus type 5 recombinant rabies glycoprotein vaccine (ONRAB)

Investigation into the genetic stability of a replication-competent human adenovirus rabies glycoprotein recombinant (ONRAB) developed for use as an oral vaccine for wildlife rabies prevention is of major importance due to the vaccine's intended placement in the environment. Using a collection of murine monoclonal antibodies directed to six distinct antigenic sites on the rabies glycoprotein, preservation of all main immunogenic epitopes of the protein after virus growth in vitro was established. A competition experiment which involved the in vitro passaging of a mixture of ONRAB and wild-type human adenovirus type 5 demonstrated that the two viruses do not exhibit noticeably different fitness levels in this environment. Nucleotide sequencing of the expression cassette of multiple viral clones recovered after 20 serial passages in cell culture and 5 serial passages in cotton rats ( Sigmodon hispidus), a species susceptible to human adenovirus infection, indicated no changes in comparison to the original virus. These trials demonstrated the stability of the insert gene of ONRAB during in vivo and in vitro passaging.

Factors Influencing The Attenuation of Serotype 1 Marek's Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication

This study was carried out to better understand factors that influenced the process of attenuation of Marek's disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and three very virulent plus (vv+) pathotype strains were passed by three techniques up to 131 times, and the passage level at attenuation was determined. The 18 attenuated or partially attenuated viruses were evaluated for protection against challenge with virulent MD virus, and the virus load (latent infection) in blood lymphocytes at 14-21 days postvaccination was determined. Viral pathotype strongly influenced the rate of attenuation. The mean passage level at attenuation for v and vv+ strains was 74 and >109, respectively. Full attenuation was achieved for nine of nine passage series with v pathotype strains but for only four of nine passage series with vv+ pathotype strains. Time to attenuation was not significantly influenced by multiplicity of infection at passage or by cell type, although a possible advantage of alternate high- and low-multiplicity passage was noted. Protection was not significantly influenced by pathotype or time to attenuation. Protection varied from 50% to 95% for the 18 passaged virus preparations; six attenuated viruses provided high protection that did not differ from that of the prototype Rispens strain. Virus load was not influenced by pathotype or by passage strategy and showed no positive correlation with protection. In several cases the most protective vaccines had the least virus load. This finding differs from previous reports and warrants further study. Variation among different strains within the same pathotype was documented for attenuation rate, protection, and virus load. Also, variation was evident when the same strain was passaged by different strategies, probably reflecting random changes during serial passage. Strain 596A (v pathotype) was the first to become attenuated, provided the best protection, and had one of the lowest virus loads. In contrast, strain 617A (v pathotype) provided the least protection and had one of the highest virus loads. Such strains provide fertile opportunities for further study.

Factors Influencing the Attenuation of Serotype 1 Marek'S Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication

Factors Influencing the Attenuation of Serotype 1 Marek'S Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication

Attenuated Foot-and-Mouth Disease Virus RNA Carrying a Deletion in the 3′ Noncoding Region Can Elicit Immunity in Swine

We constructed foot-and-mouth disease virus (FMDV) mutants bearing independent deletions of the two stem-loop structures predicted in the 3' noncoding region of viral RNA, SL1 and SL2, respectively. Deletion of SL2 was lethal for viral infectivity in cultured cells, while deletion of SL1 resulted in viruses with slower growth kinetics and downregulated replication associated with impaired negative-strand RNA synthesis. With the aim of exploring the potential of an RNA-based vaccine against foot-and-mouth disease using attenuated viral genomes, full-length chimeric O1K/C-S8 RNAs were first inoculated into pigs. Our results show that FMDV viral transcripts could generate infectious virus and induce disease in swine. In contrast, RNAs carrying the DeltaSL1 mutation on an FMDV O1K genome were innocuous for pigs but elicited a specific immune response including both humoral and cellular responses. A single inoculation with 500 microg of RNA was able to induce a neutralizing antibody response. This response could be further boosted by a second RNA injection. The presence of the DeltaSL1 mutation was confirmed in viruses isolated from serum samples of RNA-inoculated pigs or after transfection and five passages in cell culture. These findings suggest that deletion of SL1 might contribute to FMDV attenuation in swine and support the potential of RNA technology for the design of new FMDV vaccines.

Macrophage cell lines use CD81 in cell growth regulation

CD81 is an integral membrane protein belonging to the tetraspanin superfamily. It has two extracellular domains that interact with cell surface proteins and two intracellular tails that contribute to cellular processes. Although there are considerable data about how CD81 affects T- and B-cell function, not much is known about how it impacts macrophages. To address this, we established four cell lines from mouse bone marrow in the presence of macrophage colony-stimulating factor and transfection with SV40 large T antigen. Two were CD81(-/-) (ASD1 and ASD2) and two were CD81(+/-) (2ASD1.10 and 2BSD1.10). Cells were Mac-2-, PU.1-, and c-fms-positive and all the cell lines were phagocytic indicating that they were macrophage-like. In mixtures of the two cell types in tissue culture, CD81(-/-) cells out competed CD81(+/-) cells with CD81-bearing cells being undetectable after 50 cell culture passages. Although cell divisions during log-phase growth were not significantly different between CD81(+/-) macrophage cells and CD81(-/-) macrophage cells, we found that CD81(-/-) macrophage cells reached a higher density at confluency than CD81(+/-) macrophage cells. CD81 transcript levels increased as cultures became confluent, but transcript levels of other tetraspanin-related molecules remained relatively constant. Transfection of CD81 into ASD1 (CD81(-/-)) cells reduced the density of confluent cultures of transformants compared to cells transfected with vector alone. These data suggest that CD81 potentially plays a role in macrophage cell line growth regulation.

Rotarix: A Rotavirus Vaccine for the World

Single rotavirus infections have been reported to protect humans against subsequent illnesses caused by both homotypic and heterotypic rotaviruses. On the basis of these observations, a G1P[8] strain of human rotavirus named 89-12 was attenuated by multiple passages in cell culture and was developed into a vaccine candidate (herein referred to as the RIX4414 vaccine). This vaccine is currently licensed in >100 countries worldwide, many of which have a universal recommendation for all infants. The US Food and Drug Administration approved the RIX4414 vaccine for use in the United States in April 2008. This vaccine has been found to provide a reduction in the incidence of severe rotavirus disease of >80% in all trials including a developing country. No increased risk of intussusception has been associated with this vaccine, nor has the vaccine been found to interfere with responses to other routine immunizations. This article describes the history of the development of the RIX4414 vaccine.

Surveillance of Emerging Fish Viral Pathogens in Some Southeast Asian Countries

Preventing the transboundary movement of fish viral pathogens in a global environment requires active surveillance. This study examined the presence of three emerging viral pathogens among koi, common, grass, and silver carp in Cambodia, Lao PDR, Myanmar, Philippines, and Vietnam. The stud- ied viruses included koi herpesvirus (KHV), spring viremia of carp virus (SVCV), and grass carp reovirus (GCRV). Detection methods consisted of virus isolation by cell culture, infection assay in naive fish, polymerase chain reaction (PCR), and reverse-transcriptase PCR (RT-PCR). Tissues were collected and pooled from 193 fish samples in Dec. 2004 to Feb. 2005, 406 in Sep. 2005 to Feb. 2006, and 1302 in Oct. 2006 to Feb. 2007. For cell cul- ture, tissue filtrates were prepared from pooled spleens, kidneys, livers, and gills and inoculated onto koi fin (KF-1), grass carp kidney (GCK), and fat head minnow (FHM) cells. For infection assay, tissue filtrates were injected intraperitoneally to healthy, naive common carp. No virus was detected after three cell culture passages and the infection bioassays. One-step and nest- ed-step PCR was used to detect KHV in gills of fish samples. One-step and semi-nested RT-PCR was used to detect SVCV and GCRV in the spleens, kidneys, and livers of fish samples. Samples from all three years from all five countries yielded negative results for all three viruses, indicating that KHV, SVCV, and GCRV were not present in these five countries during the period of the study although KHV outbreaks had been detected in Indonesia, Taiwan, Japan, Thailand, China, and Malaysia.

A Novel Method of Dynamic Culture Surface Expansion Improves Mesenchymal Stem Cell Proliferation and Phenotype

Repeated passaging in conventional cell culture reduces pluripotency and proliferation capacity of human mesenchymal stem cells (MSC). We introduce an innovative cell culture method whereby the culture surface is dynamically enlarged during cell proliferation. This approach maintains constantly high cell density while preventing contact inhibition of growth. A highly elastic culture surface was enlarged in steps of 5% over the course of a 20-day culture period to 800% of the initial surface area. Nine weeks of dynamic expansion culture produced 10-fold more MSC compared with conventional culture, with one-third the number of trypsin passages. After 9 weeks, MSC continued to proliferate under dynamic expansion but ceased to grow in conventional culture. Dynamic expansion culture fully retained the multipotent character of MSC, which could be induced to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. Development of an undesired fibrogenic myofibroblast phenotype was suppressed. Hence, our novel method can rapidly provide the high number of autologous, multipotent, and nonfibrogenic MSC needed for successful regenerative medicine.

Essential Roles of mTOR/Akt Pathway in Aurora-A Cell Transformation

We have recently demonstrated that Aurora-A kinase is a potential oncogene to develop mammary gland tumors in mice, when expressed under MMTV promoter. These tumors contain phosphorylated forms of Akt and mTOR, suggesting that Akt-mTOR pathway is involved in transformed phenotype induced by Aurora-A. In the present studies, we discovered that stable cell lines expressing Aurora-A contain phosphorylation of Akt Ser473 after prolonged passages of cell culture, not in cells of the early period of cell culture. Levels of PTEN tumor suppressor are significantly reduced in these late passage cells at least in part due to increased poly ubiquitination of the protein. Akt-activated Aurora-A cells formed larger colonies in soft agar and are resistant to UV-induced apoptosis. Aurora-A inhibitor, VX-680, can cause cell death of Aurora-A cells in which Akt is not activated. siRNA-mediated depletion of mTOR in those cells resulted in decreased phosphorylation of Akt Ser473, suggesting that TORC2 complex phosphorylates Akt in Aurora-A cells. Treatment of late-passage Aurora-A cells with mTOR inhibitor reduced colony formation in soft agar. These results strongly suggest that commitment of cell transformation by Aurora-A is determined by at least co-activation of Akt/mTOR pathway.

Regeneration and characterization of a recombinant bovine viral diarrhea virus and determination of its efficacy to cross the bovine placenta

The capacity of different bovine viral diarrhea virus (BVDV) strains to cause transplacental infection is variable. BVDV strain SD-1 was isolated from a persistently infected heifer. Its genome represents the only reported nucleotide sequence of a noncytopathic viral isolate determined without cell culture passage in the laboratory. Thus, SD-1 might possess biological advantages over other NCP BVDV strains to be used as a model virus for investigation of viral transplacental transmission. To evaluate if a molecularly generated BVDV SD-1 is capable of crossing the bovine placenta efficiently, a full-length cDNA clone of SD-1 was constructed using RT-PCR amplification and standard molecular techniques. In vitro transcripts synthesized from the cDNA template directed the generation of infectious virus in MDBK cells with a transfection efficiency as high as 4.7 x 10(5) FFU/mug RNA. The recovered virus termed ASD1 harbored five silent point mutations engineered as genetic markers and was similar to wild type (wt) SD-1 in viral growth kinetics. As evaluated in the pregnant heifers, ASD1 was capable of crossing the bovine placenta efficiently, suggesting that NCP BVDV SD-1 is a suitable viral backbone for investigation of the role of viral genetic element(s) in viral transplacental transmission by allowing for evaluation of newly created viral mutants.