[show abstract][hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is a serious disease of children, responsible for an estimated 160,000 deaths per year worldwide. Despite the ongoing need for global prevention of RSV and decades of research, there remains no licensed vaccine. Sendai virus (SeV) is a mouse parainfluenza virus-type 1 which has been previously shown to confer protection against its human cousin, human parainfluenza virus-type 1 in African green monkeys (AGM). Here is described the study of a RSV vaccine (SeVRSV), produced by reverse genetics technology using SeV as a backbone to carry the full-length gene for RSV F. To test for immunogenicity, efficacy and safety, the vaccine was administered to AGM by intratracheal (i.t.) and intranasal (i.n.) routes. Control animals received the empty SeV vector or PBS. There were no booster immunizations. SeV and SeVRSV were cleared from the URT and LRT of vaccinated animals by day 10. Antibodies with specificities toward SeV and RSV were detected in SeVRSV primed animals as early as day ten after immunizations in both sera and nasal wash samples. One month after immunization all test and control AGM received an i.n. challenge with RSV-A2. SeVRSV-vaccinated animals exhibited reduced RSV in the URT compared to controls, and complete protection against RSV in the LRT. There were no clinically relevant adverse events associated with vaccination either before or after challenge. These data encourage advanced testing of the SeVRSV vaccine candidate in clinical trials for protection against RSV.
[show abstract][hide abstract] ABSTRACT: The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.
[show abstract][hide abstract] ABSTRACT: The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol. 82:8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK(2) cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.
Journal of Virology 03/2010; 84(6):3094-100. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.
Journal of Virology 11/2009; 84(2):810-21. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombinant Sendai virus (rSeV) in which the fusion (F) and HN surface glycoproteins (FHN) were replaced by those of hPIV-3 [rSeV(hPIV-3FHN)]. In the prophylaxis model, 129X1/SvJ mice were infected with a 90% or 20% lethal dose of the virus and were treated intranasally for 5 days with 10 mg/kg of body weight/day of either compound starting 4 h before infection. Prophylactic treatment of the mice with either compound did not prevent their death in a 90% lethality model of rSeV(hPIV-3FHN) infection. However, it significantly reduced the lung virus titers, the amount of weight lost, and the rate of mortality in mice infected with a 20% lethal virus dose. In the therapy model, mice were infected with a nonlethal dose of the virus (100 PFU/mouse) and were treated intranasally with 1 or 10 mg/kg/day of either compound for 5 days starting at 24 or 48 h postinfection. Treatment of the mice with either compound significantly reduced the virus titer in the lungs, subsequently causing a reduction in the number of immune cells and the levels of cytokines in the bronchoalveolar lavage fluid and histopathologic changes in the airways. Our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of hPIV-3 HN in our mouse model and may be promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans.
Antimicrobial Agents and Chemotherapy 07/2009; 53(9):3942-51. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSVs) are the leading causes of hospitalizations due to respiratory viral disease in infants and young children, but no vaccines are yet available. Here we describe the use of recombinant Sendai viruses (rSeVs) as candidate vaccine vectors for these respiratory viruses in a cotton rat model. Two new Sendai virus (SeV)-based hPIV-2 vaccine constructs were generated by inserting the fusion (F) gene or the hemagglutinin-neuraminidase (HN) gene from hPIV-2 into the rSeV genome. The inoculation of either vaccine into cotton rats elicited neutralizing antibodies toward both homologous and heterologous hPIV-2 virus isolates. The vaccines elicited robust and durable antibodies toward hPIV-2, and cotton rats immunized with individual or mixed vaccines were fully protected against hPIV-2 infections of the lower respiratory tract. The immune responses toward a single inoculation with rSeV vaccines were long-lasting and cotton rats were protected against viral challenge for as long as 11 months after vaccination. One inoculation with a mixture of the hPIV-2-HN-expressing construct and two additional rSeVs (expressing the F protein of RSV and the HN protein of hPIV-3) resulted in protection against challenge viruses hPIV-1, hPIV-2, hPIV-3, and RSV. Results identify SeV vectors as promising vaccine candidates for four different paramyxoviruses, each responsible for serious respiratory infections in children.
[show abstract][hide abstract] ABSTRACT: Human parainfluenza virus type 1 (hPIV-1) causes serious respiratory tract infections, especially in children. This study investigated the efficacy of the novel haemagglutinin-neuraminidase (HN) inhibitor BCX 2798 in the prophylaxis of lethal and the treatment of non-lethal parainfluenza virus infection in mice.
In the prophylaxis model, 129x1/SvJ mice were inoculated with a 90% lethal dose of a recombinant Sendai virus, in which the HN gene was replaced with that of hPIV-1 (rSeV[hPIV-1HN]). The mice were intranasally treated either once or for 5 days with 1 or 10 mg/kg/day of BCX 2798, starting 4 h before infection. In the therapeutic model, mice were infected with 100 plaque-forming units of rSeV(hPIV-1HN) per mouse and treated intranasally with 0.1, 1 or 10 mg/kg/day of BCX 2798 for 5 days, starting 24 or 48 h after infection, or for 4 days starting 72 h after infection.
Similar to multiple dosing, a single intranasal prophylaxis with 1 or 10 mg/kg of BCX 2798 protected approximately 40% or 90%, respectively, of mice from death by rSeV(hPIV-1HN) infection. BCX 2798 also significantly reduced virus lung titres (in a dose- and time-dependent manner) and reduced histopathological changes in the airways of non-lethally infected mice at multiple intranasal dosages in the therapeutic model, with the lowest effective dosage being 0.1 mg/kg/day administered 24 h after infection.
BCX 2798 was effective in the prophylaxis of lethal and in the therapy of non-lethal parainfluenza virus infection in mice, suggesting further consideration of BCX 2798 for clinical trials.
[show abstract][hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) and the human parainfluenza viruses (hPIV) are the leading causes of hospitalizations for viral respiratory tract diseases in infants and young children. Despite approximately 50 years of research, there is currently no vaccine available for any of these pathogens. Sendai virus (SeV) is a mouse respiratory virus that merits consideration as a Jennerian vaccine for hPIV-1 due to its similarity with hPIV-1 in terms of sequence, structure and antigenicity. The SeV backbone can also be manipulated using reverse genetics to create SeV-based RSV and hPIV-3 vaccines. We have prepared two recombinant SeV vaccines, expressing the fusion protein of RSV and the hemagglutinin-neuraminidase protein of hPIV-3, respectively. We found that a single intranasal vaccination with the combined recombinant SeVs (‘mixed-rSeV’) protected cotton rats from challenges with hPIV-1, hPIV-3 and RSV. This discovery, combined with our preliminary clinical demonstration that intranasal administration of unmodified SeV is safe in adults and children, makes a compelling case for advanced development of the SeV-based vaccine product.
[show abstract][hide abstract] ABSTRACT: The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-gamma-producing T cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.
[show abstract][hide abstract] ABSTRACT: BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.
Journal of Virology 07/2008; 82(17):8400-10. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The respiratory syncytial virus (RSV) is a serious pediatric pathogen for which there is currently no clinically approved vaccine. This report describes the design and testing of a new RSV vaccine construct (rSV-RSV-F), created by the recombination of an RSV F sequence with the murine parainfluenza virus-type 1 (Sendai virus, SV) genome. SV was selected as the vaccine backbone for this study, because it has previously been shown to elicit high-magnitude, durable immune activities in animal studies and has advanced to human safety trials as a xenogenic vaccine for human parainfluenza virus-type 1 (hPIV-1). Cells infected with the recombinant SV expressed RSV F protein, but F was not incorporated into progeny SV virions. When cotton rats were inoculated with the vaccine, high-titer RSV-binding and neutralizing antibodies as well as interferon-gamma-producing T-cells were induced. Most striking was the protection against intra-nasal RSV challenge conferred by the vaccine. The rSV-RSV-F construct was also tested as a mixture with a second SV construct expressing the RSV G protein, but no clear advantage was demonstrated by combining the two vaccines. As a final analysis, the efficacy of the rSV-RSV-F vaccine was tested against an array of RSV isolates. Results showed that neutralizing and protective responses were effective against RSV isolates of both A and B subtypes. Together, experimental results encourage promotion of this recombinant SV construct as a vaccine candidate for the prevention of RSV in humans.
[show abstract][hide abstract] ABSTRACT: The importance of antigen-specific CD4(+) helper T cells in virus infections is well recognized, but their possible role as direct mediators of virus clearance is less well characterized. Here we describe a recombinant Sendai virus strategy for probing the effector role(s) of CD4(+) T cells. Mice were vaccinated with DNA and vaccinia virus recombinant vectors encoding a secreted human immunodeficiency virus type 1 (HIV-1) envelope protein and then challenged with a Sendai virus carrying a homologous HIV-1 envelope gene. The primed mice showed (i) prompt homing of numerous envelope-primed CD4(+) T cell populations to the virus-infected lung, (ii) substantial production of gamma interferon, and interleukin-2 (IL-2), IL-4, and IL-5 in that site, and (iii) significantly reduced pulmonary viral load. The challenge experiments were repeated with immunoglobulin(-/-) microMT mice in the presence or absence of CD8(+) and/or CD4(+) T cells. These selectively immunodeficient mice were protected by primed CD4(+) T cells in the absence of antibody or CD8(+) T cells. Together, these results highlight the role of CD4(+) T cells as direct effectors in vivo and, because this protocol gives such a potent response, identify an outstanding experimental model for further dissecting CD4(+) T-cell-mediated immunity in the lung.
Journal of Virology 12/2007; 81(22):12535-42. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) infection is associated with secondary bacterial infections caused by nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. The pathogenesis of these complications is not completely understood; however, viral infection of respiratory epithelial cells promotes colonization by these bacteria. In the present study, RSV virions associated with NTHi and pneumococci in an inoculum-dependent manner in a fluid-phase binding assay. Adherence of NTHi and S. pneumoniae to epithelial cells transiently expressing RSV G glycoprotein was 2- and 2.2-fold higher, respectively, than adhesion to cells transfected with the vector alone (P <0.01). Furthermore, 4.6- and 6.2-fold larger numbers of NTHi and pneumococci bound to cells expressing a membrane-bound full-length RSV G protein than to cells expressing a truncated non-membrane-bound protein (P <or=0.005). Pre-incubating cells expressing membrane-bound G protein with blocking anti-RSV G antibodies reduced bacterial adherence by 78-84 % (P <or=0.005). These studies demonstrate that RSV G protein is a receptor for both NTHi and S. pneumoniae. Strategies to prevent this interaction may reduce the incidence of secondary bacterial complications of RSV infection.
Journal of Medical Microbiology 10/2007; 56(Pt 9):1133-7. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-beta into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-beta, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-beta pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV.
Virus Research 11/2006; 121(1):23-32. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-beta) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-beta completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-beta. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-beta-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-beta.
Journal of Virology 07/2006; 80(11):5145-55. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viruses of the Paramyxoviridae family are the leading cause of respiratory disease in children. The human parainfluenza viruses (hPIV) are members of the Paramyxovirinae subfamily, which also includes mumps virus, Newcastle disease virus (NDV), Sendai virus (SV) and simian type 5 virus (SV5). On the surface of these viruses is the glycoprotein hemagglutinin-neuraminidase (HN), which is responsible for cell attachment, promotion of fusion and release of progeny virions. This multifunctional nature of HN makes it an attractive target for the development of inhibitors as a treatment for childhood respiratory diseases. Here we report the crystal structure of NDV HN in complex with a derivative of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, that has a functional group designed to occupy a large conserved binding pocket around the active site. The purpose of this study was to examine the effect of a bulky hydrophobic group at the O4 position of Neu5Ac2en, given the hydrophobic nature of the binding pocket. This derivative, with a benzyl group added to the O4 position of Neu5Ac2en, has an IC(50) of approximately 10 microM in a neuraminidase assay against hPIV3 HN. The IC(50) value of the parent compound, Neu5Ac2en, in the same assay is approximately 25 microM. These results highlight the striking difference between the influenza neuraminidase and paramyxovirus HN active sites, and provide a platform for the development of improved HN inhibitors.
[show abstract][hide abstract] ABSTRACT: The enzyme neuraminidase (NA) is an attractive target for antiviral strategy because of its essential role in the pathogenicity of many respiratory viruses. NA removes sialic acid from the surface of infected cells and virus particles, thereby preventing viral self-aggregation and promoting efficient viral spread; NA also plays a role in the initial penetration of the mucosal lining of the respiratory tract. Random screening for inhibitors has identified only low-affinity and nonselective viral NA inhibitors. Selective, high-affinity inhibitors of influenza virus neuraminidase, zanamivir and oseltamivir, were developed using computer-aided design techniques on the basis of the three-dimensional structure of the influenza virus NA. These drugs were highly efficient in inhibiting replication of both influenza A and B viruses in vitro and in vivo and were approved for human use in 1999. Subsequently, the same structure-based design approach was used for the rational design of inhibitors of the parainfluenza virus hemagglutinin-neuraminidase (HN). One of these compounds, BCX 2798, effectively inhibited NA activity, cell binding, and growth of parainfluenza viruses in tissue culture and in the lungs of infected mice. Clinical reports indicate high efficiency of NA inhibitors for prophylaxis and treatment of influenza virus infection, good tolerance, and a low rate of emergence of drug-resistant mutants. Future experimental and clinical studies should establish the viability of NA inhibitors for the treatment of other respiratory virus infections.
Current Drug Targets - Infectious Disorders 01/2006; 5(4):401-9.
[show abstract][hide abstract] ABSTRACT: We have described an indirect immunoelectron microscope method to localize individual proteins on viral nucleocapsids using monoclonal antibodies and colloidal gold-conjugated second antibodies. The procedure provides good binding and retention of antibodies, good resolution(< 24nm), and negligible nonspecific binding of antibodies to the background. In addition, the method is compatible with both negative and positive staining, the two staining procedures commonly used to derive ultrastructural information on viral chromosomes. We have illustrated the procedure by localizing the NP (nucleoprotein, ≈ 2,600 copies) and P (polymerase-associated protein, ≈ 300 copies) proteins on the nucleocapsid of Sendai virus, a paramyxovirus.
Journal of Electron Microscopy Technique 02/2005; 2(2):139 - 146.