MDCK Cell Culture Research Articles

Bio‐Mechanical Properties of the Mammalian Urothelium

In the bladder urothelium, the urothelial surface umbrella cells (UC's) maintain an exceptionally tight barrier between urine and blood that prevents noxious leakage. The permeability barrier has been shown to reside on the apical surface of the umbrella cells, which are covered by specialized proteins called uroplakins. Because these proteins form plaques on the surface of umbrella cell and cover up to 90% of the urothelial surface, the urothelial surface is considered to be rigid. It is not clear how a rigid surface can be flexible as the bladder undergoes stretch and relaxation cycles. To develop an understand of the mechanical properties of the urothelial surface we attached magnetic beads coated with polylysine to the apical membrane (AM) surface of intact UC's in intact urothelia and measured their elastic moduli G, by optical magnetic tracking cytometry, OMCT. Control UC's exhibited G values which tracked closely with highly deformable red blood cells (which must deform readily to traverse capillaries). Removal of surface UC's with protamine exposed underlying intermediate cells, which exhibited G values 12–15 fold higher than UC's, and similar to cultured MDCK cells. Fixation of UC's with formaldehyde raised G by two orders of magnitude. Because submembrane actin often plays a critical role in reducing deformability, we performed confocal microscopy on control UC's and found very little subapical actin. In UC's basolateral actin was abundant, and actin was abundant under apical and basolateral membranes of intermediate cells. We conclude that UC's are strikingly deformable, despite the dense array of uroplakins in their apical membranes. Whether this deformability results from the physical effect of uroplakins or the relative lack of subapical actin will be determined using uroplakin knockout mice.

The search for new drugs synthesis and antiviral activity of derivatives of meristotropic and macedonic acids

The antiviral activity of natural triterpene acids - meristotropic and macedonic acids - and their synthetic derivatives was studied. The activities of these compounds were assessed in relation to influenza viruses A and B in MDCK cell cultures and in a model of lethal influenza pneumonia in mice. The greatest levels of in vitro activity were seen with meristotropic acid and the methyl ester of macedonic acid. Acorrelation was noted between the activities of these compounds against influenza A and B viruses. At the same time, some of the study compounds lacked in vitro activity but had protective properties in vivo, which indicates that these have some other mechanism of action not associated with actions on virus-specific targets. These results allow derivatives of meristotropic and macedonic acids to be regarded as new effective antiviral agents.

Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal transition in the embryonic heart

SUMMARYEndothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.

Attenuation of an influenza A virus due to alteration of its hemagglutinin-neuraminidase functional balance in mice

Influenza A viruses possess two surface glycoproteins, hemagglutinin (HA), which binds to sialic-acid-containing receptors, and neuraminidase (NA), which removes sialic acid from host cells. It is well established that the HA-NA functional balance regulates the efficiency of virus replication. Here, we selected a plaque variant of the WSN (H1N1) strain that grew better than the wild-type virus in NA-expressing MDCK cell culture. A reverse genetics study revealed that the single mutation HA E190K, which occurs infrequently in naturally isolated H1N1 viruses, was responsible for the phenotype of this variant. Receptor assays indicated that this mutation did not affect the receptor specificity of HA but enhanced its receptor-binding affinity, resulting in altered HA-NA functional balance relative to that of the wild-type virus. We also found that this variant replicated in nasal turbinates at an equivalent level but in lungs at a lower level compared with wild-type virus, demonstrating its attenuation in mice. Together, our data demonstrated the importance of the HA-NA functional balance for influenza virus replication in an in vivo biological setting.

Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells.

BackgroundCurrent influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines.ResultsWe evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 106 cells/mL can be infected without a cell density effect achieving titers of 1 × 108 virions/mL after 24 h.ConclusionsOverall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.

Microwave assisted synthesis and anti-influenza virus activity of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide derivatives

A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC50: 1.4μM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.

TIMP-1 Induces an EMT-Like Phenotypic Conversion in MDCK Cells Independent of Its MMP-Inhibitory Domain

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1′s ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression.

Primary cilia utilize glycoprotein-dependent adhesion mechanisms to stabilize long-lasting cilia-cilia contacts

BackgroundThe central tenet of cilia function is sensing and transmitting information. The capacity to directly contact extracellular surfaces would empower primary cilia to probe the environment for information about the nature and location of nearby surfaces. It has been well established that flagella and other motile cilia perform diverse cellular functions through adhesion. We hypothesized that mammalian primary cilia also interact with the extracellular environment through direct physical contact.MethodsWe identified cilia in rod photoreceptors and cholangiocytes in fixed mouse tissues and examined the structures that these cilia contact in vivo. We then utilized an MDCK cell culture model to characterize the nature of the contacts we observed.ResultsIn retina and liver tissue, we observed that cilia from nearby cells touch one another. Using MDCK cells, we found compelling evidence that these contacts are stable adhesions that form bridges between two cells, or networks between many cells. We examined the nature and duration of the cilia-cilia contacts and discovered primary cilia movements that facilitate cilia-cilia encounters. Stable adhesions form as the area of contact expands from a single point to a stretch of tightly bound, adjacent cilia membranes. The cilia-cilia contacts persisted for hours and were resistant to several harsh treatments such as proteases and DTT. Unlike many other cell adhesion mechanisms, calcium was not required for the formation or maintenance of cilia adhesion. However, swainsonine, which blocks maturation of N-linked glycoproteins, reduced contact formation. We propose that cellular control of adhesion maintenance is active because cilia adhesion did not prevent cell division; rather, contacts dissolved during mitosis as cilia were resorbed.ConclusionsThe demonstration that mammalian primary cilia formed prolonged, direct, physical contacts supports a novel paradigm: that mammalian primary cilia detect features of the extracellular space, not just as passive antennae, but also through direct physical contact. We present a model for the cycle of glycoprotein-dependent contact formation, maintenance, and termination, and discuss the implications for potential physiological functions of cilia-cilia contacts.

Synthesis and antiviral evaluation of C5-substituted-(1,3-diyne)-2′-deoxyuridines

Starting from acetylated 5-ethynyl-2′-deoxyuridine (3), 14 hitherto unknown C5-substituted-(1,3-diyne)-2′-deoxyuridines (with cyclopropyl, hydroxymethyl, methylcyclopentane, p-(substituted)phenyl and disubstituted-phenyl substituents) have been synthesized via a nickel-copper catalyzed C–H activation between two terminal alkynes, in yields ranging from 19% to 67%. Their antiviral activities were measured against a large number of DNA and RNA viruses including herpes simplex virus type 1 and type 2, varicella-zoster virus, human cytomegalovirus and vaccinia virus. The 5-[4-(4-trifluoromethoxyphenyl)buta-1,3-diynyl]-2′-deoxyuridine (26) is the most potent inhibitor of this series against VZV with an EC50 of ∼1 μM and a CC50 of 55 μM. Their cytostatic activities were determined against murine leukemia cells, human T-lymphocyte cells and cervix carcinoma cells. Compounds were also evaluated on a wide panel of RNA viruses, including influenza virus A (H1N1 and H3N2) and B in MDCK cell cultures, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus in Vero cell cultures and vesicular stomatitis, coxsackie B4 and respiratory syncytial virus in HeLa cell cultures and against human immunodeficiency virus type 1 and 2 in CEM cell cultures, with no specific antiviral effect. This class of compounds could be of further interest for lead optimization as anti-infectious (i.e. herpetic) agents.

The in-vitro effects of arbidol hydrochloride against 2009 new influenza virus A (H1N1)

To detect the in-vitro effects of arbidol hydrochloride against 2009 new influenza virus A (H1N1). The activity of arbidol hydrochloride against 2009 new influenza virus A (H1N1) was determined in MDCK cell cultures. Hemagglutination assay, observation of cytopathic effects, RT-PCR and quantitative RT-PCR tests were performed for determination of virus titers. Inhibition concentration 50% and cytotoxic concentration 50% were calculated with Chou's Menu of Dose-Effect Program. Arbidol hydrochloride showed low cytotoxicity (cytotoxic concentration 50%>100 μmol/L)and significant anti-2009 new influenza virus A (H1N1) activity in cell cultures. Inhibition concentration 50% were (5.5 ± 0.9), (3.4 ± 0.8), and (1.5 ± 0.2) μmol/L in hemagglutination assay, cytopathic effect test, and quantitative RT-PCR assay, respectively. Arbidol has low cytotoxicity and high anti-virus activity and can effectively trigger the activities of interferon and immune response, and therefore can be a valuable anti-influenza virus drug.

Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells

The position of the mitotic spindle plays a key role in spatial control of cell division. It is generally believed that when a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are usually unequal in size due to eccentric cleavage of the mother cell. Molecular mechanisms underlying the generation of unequal sized daughter cells have been extensively studied in Drosophila neuroblast and Caenorhabditis elegans zygote where the Gα subunit of the heterotrimeric G proteins and its binding partner – Pins in Drosophila and GPR-1/2 in C. elegans – are shown to be critical in governing spindle positioning and asymmetric cleavage of the mother cell. In mammalian system, although Gα and LGN (mammalian Pins homolog) are also required for spindle orientation, whether they can mediate asymmetric spindle positioning or asymmetric cleavage of the mother cell is not known. Here, by artificially targeting Gαi to the apical cortex in 3-D cultured MDCK cells, we established a system where asymmetric spindle positioning can be consistently induced. Interestingly, this asymmetrically positioned spindle does not lead to asymmetric cleavage; instead it results in equal sized daughter cells. Live cell time-lapse analysis revealed that anaphase spindle elongation compensated the original asymmetric spindle positioning. Our findings demonstrate that asymmetric spindle positioning does not necessarily lead to unequal sized daughter cells in mammalian system. We discuss potential mechanisms in generating unequal sized daughter cells.

Novel, Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×102 to 5.50×107 copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.

Cytotoxicity of Southeast Asian snake venoms

Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus) was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50) under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL) and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL) whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL) against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes.

Amoebic liver abscess production by Entamoeba dispar

Although Entamoeba dispar displays a similar morphology to Entamoeba histolytica, cellular and molecular studies have revealed significant differences between these two amoebae, including the former being characterized as non-pathogenic and the later as pathogenic. However, recent in vivo and in vitro experiments have shown that E. dispar strains of different origin are capable of causing liver damage and destroying cell culture lines in the presence of common intestinal bacteria. These results suggested that E. dispar may present pathogenic behavior according to the specific E. dispar strain, culture and environmental conditions. To investigate this possibility, we carried out in vivo and in vitro studies using a xenic strain E. dispar (ICB-ADO) isolated from a symptomatic non-dysenteric Brazilian patient. This strain was able to induce liver necrosis in a hamster model that was more severe than that produced by E. histolytica. The ICB-ADO isolate also caused significantly more destruction of cultured MDCK cells and increased loss of transepithelial resistance than did the E. histolytica. Xenic E. dispar exhibited high proteolytic activity, which was partially inhibited by the addition of cysteine-protease inhibitors. Based on our biochemical and molecular characterization of E. dispar (ICB-ADO) xenic culture and its ability to produce liver abscesses, we conclude that this specific strain can indeed produce tissue damage, distinct from the frequently used non-pathogenic E. dispar SAW 760 strain.

Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology (96%-99%) with known epidemic strains (A/California/04/2009(H1N1). Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences: A/Hubei/86/2009 PKVRDQEG → PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG → PKVRDQER, A/Hubei/75/2009 PKVRDQEG → PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein, 627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

Optimization of roller culturing of influenza virus vaccine strains in MDCK and Vero cell cultures using plant origin components

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).

Synthesis and antiviral activity of functionally substituted indole-3-acetic acids

Methods for the synthesis of dibromo-substituted derivatives of indomethacin and related N-alkylated indole-3-acetic acid derivatives have been developed. Pathways for the introduction of various substituents, in particular nitrogen-containing groups, into the structure of auxins have been studied. The antiviral activity of some brominated functionally substituted compounds with respect to A/Aichi/2/69 influenza virus in MDCK cell culture and an influenza pneumonia model in mice has been evaluated.

Synthesis and anti-influenza activity of synthetic ribonucleases

The acute toxicity and antiviral activity of synthetic ribonucleases with various structures have been studied. It is established that synthetic ribonucleases exhibit low toxicity with respect to MDCK cell cultures and inhibit to different extents the reproduction of influenza A and B type viruses.

Analysis on the etiological surveillance of influenza/novel influenza A (H1N1) from 2009-2010 in Shanxi Province

To study the epidemical characteristics of influenza/novel influenza A (H1N1) in Shanxi province from 2009 to 2010, and to provide scientific foundations for predicting and controlling the pandemic outbreak of influenza/novel influenza A (H1N1) effectively. All samples were collected from cases that resemble influenza cases in sentinel hospital and influenza outbreak. The influenza were detected by PCR and isolated by MDCK cell culture method. Finally, Shanxi province surveillance data from May, 2009 to April, 2010 of influenza like illness (ILI) cases and pathogen detections were analyzed. In Shanxi province, influenza viruses kept activation in whole year. The predominant pandemic strain in 2009 was novel influenza A (H1N1) virus. The strong peak was around November, 2009 [positive rate: 58.1%, novel influenza A ( H1N1) of the total: 88.1%]. As well, the people infected influenza caused by novel influenza A (H1N1) were mainly under 59-year-old, and the higher positive rates were concentrated in the people from 5-year-old to 24-year-old. In 2010, influenza B (Victoria) viruses were mainly detected from clinical specimens and became the dominant strain. Surveillance of Influenza liue illnes, (ILI) and etiology, which can promptly reflect the influenza epidemic situation, play a significant role for understanding epidemic rule of influenza/novel influenza A (H1N1).

Construction of a recombinant bacmid DNA in order to express Neuraminidase in insect cell line

Non-replicating virus-like particles (VLPs) have been suggested as a promising platform for viral vaccines. Recently several studies have introduced VLPs produced in insect cells as a vaccine candidate against influenza infection. Neuraminidase (NA), one of the two membrane glycoproteins of influenza A viruses has been employed to construct such influenza VLPs to induce protective immunity. In this study, the human influenza virus (A /New Caledonia 20/1999/ (H1N1)) was propagated in MDCK cell culture. Viral RNA was extracted using RNX-plus solution. Complementary DNA synthesis was carried out using uni-12 primer. NA open reading frame (1413 bp) was amplified by RT-PCR using high fidelity Taq DNA polymerase. The amplicon was purified from the gel and cloned into pFastBac1 plasmid through SalI/XhoI sites. After verification of cloned NA by restriction analysis, it was subjected to automated sequencing bidirectionally. The recombinant pFastBacNA vector was transformed to E.coli DH10Bac cells which harbor bacmid DNA and helper plasmid to create NA recombinant bacmid. Restriction map analysis and sequencing results confirmed the fidelity of NA sequence. Homologous recombination between pFastBacNA and bacmid and creation of NA recombinant bacmid was verified by PCR using NA specific and M13 universal primers. In an ongoing study, the cultured Sf9 (Spodoptera frugiperda) insect cell line will be transfected with NA recombinant bacmid to produce recombinant baculovirus expressing NA gene. This production is in its native highly glycosylated form appropriate to use in vaccine research projects. Recombinant baculovirus expressing NA gene can be also used with other individual recombinant baculoviruses expressing HA and M1 genes in production of influenza VLPs.