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JOURNAL OF VIROLOGY, Nov. 2010, p. 10999–11009
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 21
Ultrastructural and Biophysical Characterization of Hepatitis
C Virus Particles Produced in Cell Culture?
Pablo Gastaminza,1*† Kelly A. Dryden,2† Bryan Boyd,1Malcolm R. Wood,3
Mansun Law,1Mark Yeager,2,4,5and Francis V. Chisari1
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 920371; Department of
Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-08862;
TSRI Core Microscopy, The Scripps Research Institute, La Jolla, California 920373; Department of Cell and
Molecular Biology, The Scripps Research Institute, La Jolla, California 920374; and Division of
Cardiovascular Diseases, Scripps Clinic, La Jolla, California 920375
Received 10 March 2010/Accepted 18 July 2010
We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced
in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (?40- to 75-nm
diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their
surfaces. Electron cryomicroscopy revealed two major particle populations of ?60 and ?45 nm in diameter.
The ?60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially
separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed
a high infectivity-to-HCV RNA ratio. The ?45-nm particles lacked a membrane bilayer and displayed a higher
buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-
density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution
ultrastructural information of particle populations displaying differential biophysical properties and specific
infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV
RNA, and viral antigens suggests that infectious particles are likely to be present in the large ?60-nm HCV
particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understand-
ing the structural characteristics of infectious HCV particles.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis
worldwide, with approximately 170 million humans chronically
infected. Persistent HCV infection often leads to fibrosis, cir-
rhosis, and hepatocellular carcinoma (27). There is no vaccine
against HCV, and the most widely used therapy involves the
administration of type I interferon (IFN-?2?) combined with
ribavirin. However, this treatment is often associated with se-
vere adverse effects and is often ineffective (53).
HCV is a member of the Flaviviridae family and is the sole
member of the genus Hepacivirus (43). HCV is an enveloped
virus with a single-strand positive RNA genome that encodes a
unique polyprotein of ?3,000 amino acids (14, 15). A single
open reading frame is flanked by untranslated regions (UTRs),
the 5? UTR and 3? UTR, that contain RNA sequences essen-
tial for RNA translation and replication, respectively (17, 18,
26). Translation of the single open reading frame is driven by
an internal ribosomal entry site (IRES) sequence residing
within the 5? UTR (26). The resulting polyprotein is processed
by cellular and viral proteases into its individual components
(reviewed in reference 55). The E1, E2, and core structural
proteins are required for particle formation (5, 6) but not for
viral RNA replication or translation (7, 40). These processes
are mediated by the nonstructural (NS) proteins NS3,
NS4A, NS4B, NS5A, and NS5B, which constitute the mini-
mal viral components necessary for efficient viral RNA rep-
lication (7, 40).
Expression of the viral polyprotein leads to the formation of
virus-like particles (VLPs) in HeLa (48) and Huh-7 cells (23).
Furthermore, overexpression of core, E1, and E2 is sufficient
for the formation of VLPs in insect cells (3, 4). In the context
of a viral infection, the viral structural proteins (65), p7 (31, 49,
61), and all of the nonstructural proteins (2, 29, 32, 41, 44, 63,
67) are required for the production of infectious particles,
independent of their role in HCV RNA replication. It is not
known whether the nonstructural proteins are incorporated
into infectious virions.
The current model for HCV morphogenesis proposes that
the core protein encapsidates the viral genome in areas where
endoplasmic reticulum (ER) cisternae are in contact with lipid
droplets (47), forming HCV RNA-containing particles that
acquire the viral envelope by budding through the ER mem-
brane (59). We along with others showed recently that infec-
tious particle assembly requires microsomal transfer protein
(MTP) activity and apolipoprotein B (apoB) (19, 28, 50), sug-
gesting that these two components of the very-low-density li-
poprotein (VLDL) biosynthetic machinery are essential for the
formation of infectious HCV particles. This idea is supported
by the reduced production of infectious HCV particles in cells
that express short hairpin RNAs (shRNAs) targeting apoli-
poprotein E (apoE) (12, 30).
HCV RNA displays various density profiles, depending on
the stage of the infection at which the sample is obtained (11,
58). The differences in densities and infectivities have been
* Corresponding author. Present address: Department of Cellular
and Molecular Biology, Centro Nacional de Biotecnologia (CNB-
CSIC), Darwin 3, Madrid 28049, Spain. Phone: 34 915 854 4561. Fax:
34 915 854 506. E-mail: firstname.lastname@example.com.
† P.G. and K.D. contributed equally to this work.
?Published ahead of print on 4 August 2010.
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attributed to the presence of host lipoproteins and antibodies
bound to the circulating viral particles (24, 58). In patients,
HCV immune complexes that have been purified by protein A
affinity chromatography contain HCV RNA, core protein, tri-
glycerides, apoB (1), and apoE (51), suggesting that these host
factors are components of circulating HCV particles in vivo.
Recent studies using infectious molecular clones showed
that both host and viral factors can influence the density profile
of infectious HCV particles. For example, the mean particle
density is reduced by passage of cell culture-grown virus
through chimpanzees and chimeric mice whose livers contain
human hepatocytes (39). It has also been shown that a point
mutation in the viral envelope protein E2 (G451R) increases
the mean density and specific infectivity of JFH-1 mutants (70).
HCV particles exist as a mixture of infectious and noninfec-
tious particles in ratios ranging from 1:100 to 1:1,000, both in
vivo (10) and in cell culture (38, 69). Extracellular infectious
HCV particles have a lower average density than their nonin-
fectious counterparts (20, 24, 38). Equilibrium sedimentation
analysis indicates that particles with a buoyant density of ?1.10
to 1.14 g/ml display the highest ratio of infectivity per genome
equivalent (GE) both in cell culture (20, 21, 38) and in vivo (8).
These results indicate that these samples contain relatively
more infectious particles than any other particle population.
Interestingly, mutant viruses bearing the G451R E2 mutation
display an increased infectivity-HCV RNA ratio only in frac-
tions with a density of ?1.1 g/ml (21), reinforcing the notion
that this population is selectively enriched in infectious parti-
The size of infectious HCV particles has been estimated in
vivo by filtration (50 to 80 nm) (9, 22) and by rate-zonal cen-
trifugation (54 nm) (51) and in cell culture by calculation of the
Stokes radius inferred from the sedimentation velocity of in-
fectious JFH-1 particles (65 to 70 nm) (20). Previous ultra-
structural studies using patient-derived material report parti-
cles with heterogeneous diameters ranging from 35 to 100 nm
(33, 37, 42, 57, 64). Cell culture-derived particles appear to
display a diameter within that range (?55 nm) (65, 68).
In this study we exploited the increased growth capacity of a
cell culture-adapted virus bearing the G451R mutation in E2
(70) and the enhanced particle production of the hyperpermis-
sive Huh-7 cell subclone Huh-7.5.1 clone 2 (Huh-7.5.1c2) (54)
to produce quantities of infectious HCV particles that were
sufficient for electron cryomicroscopy (cryoEM) analyses.
These studies revealed two major particle populations with
diameters of ?60 and ?45 nm. The larger-diameter particles
were distinguished by the presence of a membrane bilayer,
characterized by electron density attributed to the lipid head-
groups in its leaflets. Isopycnic ultracentrifugation showed that
the ?60-nm particles are enriched in fractions with a density of
?1.1 g/ml, where optimal infectivity-HCV RNA ratios are ob-
served. These results indicate that the predominant morphol-
ogy of the infectious HCV particle is spherical and pleomor-
phic and surrounded by a membrane envelope.
Cells and viruses. Hyperpermissive Huh-7.5.1 cell subclone Huh-7.5.1 clone 2
cells (54, 69) were cultured in Dulbecco's modified Eagle's medium (DMEM)–
10% fetal calf serum (FCS) at 37°C and 5% CO2. Cell culture-adapted JFH-1
variant D183 virus was previously described (70).
Determination of viral infectivity titers and HCV RNA copy number. Infec-
tivity titers were determined in Huh-7 cells by endpoint dilution and immuno-
fluorescence, as previously described (20, 69). Infectivity titers are expressed as
the number of infection focus forming units per ml (FFU/ml).
HCV RNA levels were determined in total cellular RNA extracted by the
guanidinium thiocyanate-phenol-chloroform (GTC) method (13) followed by
quantitative reverse transcription-PCR (RT-qPCR), as previously described
(69). To determine HCV copy numbers, standard curves were prepared by serial
10-fold dilution of a known amount of a plasmid bearing the amplified HCV
sequence (69).
Virus production, concentration, and purification. Huh-7.5.1. cells (3.5 ? 106
cells/flask) were plated in T162 vented cap cell culture flasks (Corning, Lowell,
MA). The next day, cells were inoculated at a multiplicity of 0.01 with D183 virus
stocks and incubated at 37°C. Supernatants were collected after 3 days and
replaced with 15 ml/flask of serum-free OptiMem medium (Invitrogen, Carlsbad,
CA). After an additional 20 h of incubation, the supernatants were collected, the
cells were replenished with 15 ml of warm OptiMem and incubated for an
additional 20 h, and then the final supernatants were collected. The supernatants
from days 4 and 5 were filtered through a 0.45-?m-pore-size filter and stored at
Crude virus supernatants, typically ?300 ml (from 20 T162 flasks), were
concentrated in Centricon centrifugal devices with a 100-kDa cutoff (Millipore,
Temecula, CA) by centrifugation at 3,500 rpm until they reached the desired
final volume (?1 ml). Concentrated samples were overlaid onto a 1-ml 20%
sucrose-TNE (10 mM Tris-HCl, pH 8, 150 mM NaCl, 2 mM EDTA) layer
floating over a cushion of 1 ml of 60% sucrose-TNE (Fig. 1A). Samples were
subjected to ultracentrifugation in an SW60 rotor for 2 h at 150,000 ? g. Four
fractions were collected by aspiration from the top, and most of the infectivity
and HCV RNA were concentrated in fraction 3, which corresponded to the 20 to
60% sucrose interphase (Fig. 1A). Sucrose was removed from fraction 3 by
exchange into TNE buffer by centrifugation in Microcon-100 centrifugal devices
with a 100-kDa cutoff (Millipore, Temecula, CA).
Purification of total virus populations by sedimentation velocity. Cushion-
purified virus samples were diluted in 200 ?l of TNE buffer and overlaid onto a
continuous (10 to 50%) sucrose gradient (3.6 ml) (Fig. 1B). Rate-zonal ultra-
FIG. 1. Infectious HCV particles purified by sucrose gradient cen-
trifugation. (A) Concentrated supernatants isolated from infected cells
were subjected to ultracentrifugation on a sucrose step gradient.
(B) The particles collected from the 20–60% interface (fraction 3)
were further purified in a continuous 10 to 50% sucrose gradient.
Infectivity and HCV RNA levels were determined by titration and
quantitative PCR, and the results were expressed as the numbers of
FFU per ml and HCV RNA genome equivalents (GE) per ml,
Page 3
centrifugation at 200,000 ? g for 1 h separated particles in the supernatant by
virtue of their sedimentation velocity (20). Twelve fractions (?300 ?l) were
collected from the top and analyzed for viral infectivity and HCV RNA. Most of
the infectivity and HCV RNA were typically located in fractions 5 and 6 (Fig.
1B), which were pooled, buffer exchanged into phosphate-buffered saline (PBS),
and concentrated into small volumes (?25 ?l) by ultrafiltration through Micro-
con-100 filters (Millipore, Temecula, CA).
Purification of viral particles by isopycnic ultracentrifugation density. Cush-
ion-purified, concentrated virus samples (?200 ?l) were overlaid onto a discon-
tinuous gradient with 20, 30, 40, 50, and 60% sucrose steps (?700 ?l) and
subjected to overnight ultracentrifugation at 120,000 ? g, as previously described
(20). Twelve fractions were collected from the top and analyzed for buoyant
density, viral infectivity, and HCV RNA content. The highest infectivity-HCV
RNA ratio was detected in fractions whose density ranged from 1.10 to 1.14 g/ml.
These fractions were pooled, buffer exchanged into PBS using Microcon filters as
described above, and compared with pooled fractions of lower (?1.1g/ml) and
higher (?1.4 g/ml) densities, which displayed substantially lower specific infec-
tivity than the intermediate-density fractions.
Virus immunoprecipitation experiments. Agarose beads (?50 ?l) containing
the monoclonal neutralizing recombinant human IgG against HCV E2 (AR3A)
(36) or the anti-HIV recombinant human IgG (B6) (52) were kindly provided by
Mansun Law (The Scripps Research Institute, La Jolla, CA). The beads were
equilibrated in PBS and incubated overnight at 4°C with ?250 ?l of crude virus
preparations in OptiMem (diluted 1:5 in PBS). As a control, diluted virus was
incubated in the absence of beads to determine the baseline infectivity and HCV
RNA values. After overnight incubation, the supernatant was collected and
tested for infectivity. RNA was extracted from washed (three times with PBS)
beads using GTC (4.2 M guanidine thiocyanate, 0.75 M sodium citrate, pH 7.3,
0.5% sarcosyl) (13), and the HCV RNA copy number was determined as de-
scribed above.
Negative-stain electron microscopy. Samples (25 ?l) were fixed by addition of
1 volume of 8% paraformaldehyde (PFA) in 0.1 M cacodylate buffer (pH 7.4).
After incubation for 20 min, aliquots (4 to 5 ?l) were placed on Parafilm (Alcan
Packaging-Neenah, WI). Parlodion-coated nickel grids were subjected to glow
discharge and then immediately inverted onto the droplets. After incubation for
3 min at room temperature (RT), the grids were blotted, and negative staining
was performed by application of 3% uranyl acetate (pH 4) for 2 min at RT. The
stain was removed by blotting, and the grids were air dried. Grids were examined
using a transmission electron microscope (Philips FEI CM100; Eindhoven, Neth-
erlands) operating at 100 kV. Digital images were generated by direct recording
using a charge-coupled-device (CCD) camera (SIS Megaview III camera; Olym-
pus, Munster, Germany) or by scanning micrographs (Finescan 2750 high reso-
lution scanner; Fujifilm, Edison, NJ).
Immunolabeling. Sample grids were inverted onto a PBS–5% bovine serum
albumin (BSA) droplet for 5 min at RT and then incubated for 1 h at RT with
human monoclonal anti-E2 AR3A antibody (36) and/or a mouse monoclonal
anti-apoE antibody (clone 3D12; Meridian Life Science, Inc., Memphis, TN)
diluted 1:20 and 1:10 in PBS–1% BSA, respectively. The grids were then washed
three times (2 min each time) with PBS–1% BSA, followed by incubation for 1 h
at RT with the corresponding secondary antibodies conjugated to 6- or 12-nm
gold particles (Jackson Labs, Watson Grove, PA) at various dilutions in PBS–1%
BSA. The grids were then washed with PBS, fixed for 5 min in PBS–1% glutar-
aldehyde, washed with H2O for 5 min, and stained and imaged as described
Detergent treatment. Purified particles were fixed with 1% glutaraldehyde for
20 min at RT. One aliquot was treated with one volume of 0.4% NP-40 in PBS
for 5 min, and a second aliquot was treated with detergent-free PBS. Both
samples were then purified through a 20% sucrose cushion, buffer exchanged
into PBS, and then negatively stained as described above.
Viral antigen quantification. Relative core protein levels were determined by
enzyme-linked immunosorbent assay (ELISA) as previously described (20). Rel-
ative E2 levels were quantified by ELISA using a recombinant anti-E2 mono-
clonal IgG (AR3A) (36). Briefly, gradient fractions were adsorbed overnight at
4°C onto a Nunc Maxisorp ELISA plate (Thermo Fisher Scientific, Rochester,
NY). Wells were washed twice with 200 ?l of PBS and fixed for 20 min at RT with
4% PFA in PBS (pH 7). Wells were washed twice and blocked with 5% nonfat
milk in PBS for 1 h at RT. Wells were then washed twice with 200 ?l of PBS and
replenished with 50 ?l of a 2 ?g/ml dilution of the recombinant anti-E2 IgG
(AR3A) (36) in binding buffer (3% BSA–0.3% Triton X-100 in PBS). Wells were
washed four times with 200 ?l of PBS and replenished with 50 ?l of an 80 ng/ml
dilution of horseradish peroxidase (HRP)-conjugated goat anti-human antibod-
ies (Pierce, Rockford, IL). After incubation for 1 h at RT, wells were washed four
times with PBS before the developing reagent was added (Pierce, Rockford, IL).
The colorimetric reaction was stopped by adding 1 volume of 1 M H2SO4. Serial
2-fold dilutions of the peak fraction were used to generate a standard curve that
was used for conversion of the values of the optical density 450 nm (OD450).
Electron cryomicroscopy. For biosafety, highly concentrated virus prepara-
tions were fixed in PFA-cacodylate buffer as described above to inactivate viral
infectivity. Purified HCV samples were vitrified by standard methods for cryoEM
(66). In brief, an aliquot (?3 ?l) was applied to a glow-discharged, perforated
carbon-coated grid (either 2/4 Cu-Rh Quantifoil or 2/2-4C C-flat), blotted with
filter paper, and rapidly plunged into liquid ethane. Low-dose images were
recorded at a magnification of ?50,000 on an FEI Tecnai F20 Twin transmission
electron microscope operating at 120 kV, with a nominal underfocus ranging
from 1.5 to 3.5 ?m and a pixel size of 0.271 nm at the specimen level. All images
were recorded with a Gatan 4,000- by 4,000-pixel CCD camera utilizing the
manual mode of Leginon data collection software (62). The grids were main-
tained at ?180°C using a Gatan 626 cryo-stage.
Image analysis. Image analysis was performed using the NIH open source
software Image J (available at: http://rsbweb.nih.gov/ij/). Particle diameters were
measured on digitized images, both point to point by ruler and by comparison
with circles of defined radii. Analysis was performed only on intact particles, with
fewer than 1% of the particles appearing to be broken.
Classification of particle types into zero, one, or multiple membrane bilayers
was based on morphological analysis of the cryoEM images by two or more
independent observers. Only particles displaying a distinct electron-dense bilayer
were considered enveloped (E). Nonenveloped (NE) particles did not display a
visible bilayer. A minor population of large vesicular (LV) particles was observed
that consisted of enveloped particles whose diameters were larger than 85 nm.
Finally, a minor multivesicular (MV) particle population was observed that
displayed two or more clearly distinguishable bilayers.
For illustration, raw images were resized and processed using the Gaussian
blur algorithm (radius, 2 to 3 pixels) in Adobe Photoshop CS2 (version 9.0.2)
for Mac.
Statistical analysis. Statistical significance of the difference between the mean
diameters of particles with different buoyant densities was calculated using a one
tailed, two-sample t test assuming unequal variance (heteroscedastic).
Preparation of highly purified HCV particles. The low yield
of infectious HCV in cell culture has impeded ultrastructural
and biochemical analysis. This barrier has been partially over-
come by the development of a robust cell culture system. By
the use of a cell culture-adapted (D183) virus with vigorous
growth characteristics (70) and a hyperpermissive cell clone
derived from Huh-7.5.1 cells (54), the yield of infectious virus
was increased by two logs from a titer of ?104to ?106FFU/
ml. Infection of Huh-7.5.1 clone 2 (Huh-7.5.1 c2) cells at a low
multiplicity of infection ([MOI] 0.01) produced stocks that
routinely contained 2 ? 106to 5 ? 106FFU/ml 3 days after
infection. Substitution of complete medium with serum-free
OptiMem (Invitrogen, Carlsbad, CA) 20 h before collection of
the supernatants did not reduce the yield of infectious virus
(data not shown), and high-titer (?106FFU/ml) virus stocks
with reduced serum content could be generated. This enabled
efficient concentration of infectious supernatants by ultrafiltra-
tion through anisotropic membranes (Centricon filters with a
100-kDa cutoff; Millipore, Temecula, CA), resulting in virus
preparations containing ?108FFU/ml and nearly 1011HCV
RNA GEs/ml.
We purified the concentrated viral particles by ultracentrif-
ugation as described in Materials and Methods. Briefly, con-
centrated supernatants were pelleted through a 20% sucrose
cushion onto a 60% cushion (Fig. 1A). Partially purified par-
ticles were collected from the 20–60% interface, where most
(?75%) of the infectivity and HCV RNA were recovered (Fig.
1A, fraction 3). This material was subjected to ultracentrifu-
gation in a continuous 10 to 50% sucrose gradient to purify
VOL. 84, 2010 cryoEM ULTRASTRUCTURE OF HCV PARTICLES11001
Page 4
viral particles based on their sedimentation velocities (Fig. 1B).
This procedure permitted recovery of virtually all the infectiv-
ity (?85%) and HCV RNA in a single peak (Fig. 1B, fraction
5), which migrated with the expected sedimentation velocity
(20). In contrast to what we observed for the parental JFH-1
virus (20), the majority of both the infectivity and the HCV
RNA of the D183 virus comigrated in the same fraction (Fig.
1B, fraction 5), probably due to the higher average density of
the infectious D183 virus particles reflecting a point mutation
(G451R) in the viral E2 protein (70).
By negative-stain EM, virus-like particles (VLPs) were ob-
served only in the samples from infected cells (Fig. 2) and not
in parallel, control samples from noninfected cells (data not
shown). These preparations revealed a pleomorphic particle
population with various diameters (58.7 ? 19.3 nm; n ? 450)
(Fig. 2), similar to those previously described for JFH-1 virus
produced in cell culture (23, 65) and from infected patients
(42, 57, 64). The particles displayed a smooth surface with no
visible surface projections (Fig. 2).
In order to confirm that these preparations contained HCV
particles, we incubated them with a well-characterized E2-
specific neutralizing antibody (AR3A) (36) known to specifi-
cally immuno-deplete viral infectivity and immunoprecipitate
HCV RNA from infectious supernatants (see Fig. S1 posted at
A small but significant fraction of the particles (5 to 20%)
displayed specific decoration with 12-nm gold-conjugated sec-
ondary antibody only after incubation with anti-E2 antibodies
(Fig. 2C). It is noteworthy that the antibody-labeled particles
were morphologically indistinguishable from those not deco-
rated (see Fig. S2 posted at http://www.scripps.edu/wieland
/data/Gastaminza/FigureS2.tif). No decoration was observed
with isotype control antibody (data not shown) directed against
the HIV envelope (52), confirming the specificity of the inter-
action and the viral nature of the purified particles. Since we
failed to specifically decorate the particles with different anti-
bodies against the core protein, the major structural compo-
nent of HCV particles, we set out to determine if the purified
particles could be specifically decorated with antibodies against
apolipoprotein E, a cellular factor shown to be associated with
HCV particles in vivo (51) and in vitro (12). Virus-like particles
could be specifically decorated with anti-apoE (12-nm gold)
(Fig. 2D) and anti-E2 (6 nm-gold) (Fig. 2D, white arrows) and
not with an isotype control antibody, reinforcing the notion
that the purified particles display antigens previously found in
HCV virions.
When the virus preparation was treated with detergent
(0.2% NP-40), all of the infectivity and most (?80%) of the
HCV RNA content were lost (Fig. 3A), suggesting that the
detergent removed the envelope from the virions and destabi-
lized most of the capsids (46) (Fig. 3A). Negative-stain EM
showed that the detergent-treated particles were smaller in
diameter (45 ? 5 nm; n ? 83) than untreated particles (56.1 ?
10 nm; n ? 145) (Fig. 3B), which recapitulated the decrease in
Stokes radii calculated by sedimentation velocity for patient-
derived particles before and after detergent treatment (51).
Detergent-treated particles also displayed a rough, angular
surface (Fig. 3B, inset) that contrasts with the relatively
smooth surface observed in untreated particles (Fig. 3B, inset).
Negatively stained particles were pleomorphic with an aver-
age diameter of ?55 nm. However, the use of heavy metal
stain (uranyl acetate), a solid support (carbon), and subse-
quent drying would likely result in deformation and loss of
native structure. Thus, we examined the virus preparations by
electron cryomicroscopy, where particles are vitrified in solu-
tion and observed in a close-to-native state.
cryoEM of purified particles. Frozen hydrated particles (Fig.
4) were pleomorphic and similar in appearance to the nega-
tively stained particles (Fig. 2), with an average diameter of
54.6 ? 12.6 nm (range, 31 to ?100 nm; n ? 2,087). The
predominant morphology was spherical, with a relatively
smooth surface devoid of visible projections (Fig. 4). cryoEM
revealed that many of the particles displayed a 5- to 6-nm-thick
electron-dense bilayer (Fig. 4, arrowheads), compatible with a
lipid membrane (25). This feature was used to differentiate the
particle populations into four distinct classes (Fig. 5). Particles
that displayed a visible bilayer were designated enveloped (E),
and particles devoid of this feature were designated nonenvel-
oped (NE) (Fig. 5A). A small number of large vesicles dis-
played one bilayer (LV), and multivesicular particles (MV)
displayed multiple bilayers.
The enveloped and nonenveloped populations were most
abundant in these preparations (94%) and had similar distri-
butions (E, 46%; NE, 53%) while the LV and MV particles
represented only 4% and 1.7%, respectively. Importantly, the
E particles displayed smooth surfaces, and some displayed
clearly discernible internal structures that were separated from
the lipid bilayers. This capsid-like object (Fig. 5) was similar in
size to the NE particles, which were compact and electron
dense, devoid of any internal features or surface projections
(Fig. 5A). LV particles were electron lucent and occasionally
displayed surface projections (Fig. 5A) while MV particles
displayed variable sizes and morphologies and contained het-
erogeneous structures, including internal vesicles and amor-
phous material (Fig. 5A).
These four types of particles displayed different mean diam-
eters (Fig. 5B). The E particles (n ? 880) had a mean diameter
of 60.3 ? 10.4 nm. In contrast, the NE particles had a smaller
diameter with a narrower size distribution (44.24 ? 4.74 nm;
n ? 1,089). Finally, LV and MV particles were much larger
(?85 nm), with extremely variable diameters of 105.6 ? 22.4
nm (n ? 82) and 114.9 ? 31.8 nm (n ? 36), respectively. Four
different virus preparations displayed similar frequencies and
size distributions of the four different particle populations
(see Fig. S3 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS3.tif), which attests to the consistency of
the purification method and analysis.
cryoEM, infectivity, and viral RNA analysis of particles of
different buoyant densities. The previous results show that the
predominant E and NE particle classes isolated from infection
supernatants were associated with most of the HCV RNA and
infectivity. To confirm the viral nature of these particles, we
modified the purification scheme to separate the different par-
ticles based on their buoyant densities. Cushion-purified par-
ticles were separated by ultracentrifugation in isopycnic su-
crose density gradients. Infectivity, HCV RNA, envelope (E2
protein), core protein content, and density were determined
for each fraction as described in Materials and Methods and
Gastaminza et al. (20). The G451R mutation is known to
produce a distinct increase in the average density of infectious
Page 5
D183 virus particles relative to the parental JFH-1 virus (70).
As expected for the D183 virus, infectivity and HCV RNA
were most abundant in fractions whose density was ?1.15 g/ml
(Fig. 6A, fractions 8 and 9). Also as expected from previous
experiments (16, 20, 21), fractions around 1.1 g/ml (Fig. 6A,
fractions 6 and 7) that preceded the peak of HCV RNA and
infectivity consistently displayed the highest infectivity-HCV
RNA ratio, suggesting that they were relatively enriched in
FIG. 2. HCV particles are pleomorphic and display E2 and apolipoprotein E on their surface. (A) Electron micrograph of highly purified,
negatively stained HCV particles demonstrates their fairly uniform size but pleomorphic shape. Particles were purified as described in the legend
of Fig. 1. Scale bar, 500 nm. (B) Close-up views show the heterogeneous sizes and shapes. (C) Representative images show specific decoration of
a subset (5 to 20%) of particles with an antibody directed against the viral envelope protein E2. (D) Representative images show specific decoration
of E2-positive particles (6-nm gold) with anti-apoE antibodies (12-nm gold). Scale bar, 100 nm (B, C, and D).
VOL. 84, 2010 cryoEM ULTRASTRUCTURE OF HCV PARTICLES11003
Page 6
infectious versus noninfectious particles (Fig. 6B; see also Fig.
S4 posted at http://www.scripps.edu/wieland/data/Gastaminza
/FigureS4.tif). Therefore, the intermediate fractions preceding
the peak of infectivity likely contain the largest proportion of
infectious particles, albeit in lower numbers.
cryoEM was then used to evaluate particle morphology in
three density pools. The low-density pool (density of ?1.1g/ml)
(low specific infectivity) typically contained less than 2% of the
infectivity and HCV RNA of the preparation. The intermedi-
ate-density pool (?1.10 to 1.14 g/ml) displayed the highest
specific infectivity and contained ?15% of the infectivity and
5% of the total HCV RNA. Finally, the high-density pool
(density of ?1.14g/ml) (low specific infectivity) contained most
of the infectivity (?80%) and HCV RNA (?94%) and most of
the virus-like particles.
As shown in Fig. 6C, the low-density pool that had a low
specific infectivity contained a mixture of E, LV, and MV
particles, with no particular class dominating the population.
In contrast, the intermediate-density pool that had a high spe-
cific infectivity was greatly enriched in E particles, which con-
stituted 49% of the total particle population. The NE, LV, and
MV particles were relatively underrepresented (20, 24, and
6%, respectively). Finally, NE particles dominated the high-
density pool that had a low specific infectivity, representing
85% of the particles in that population, while E particles were
much less frequent (15%). The correlation between the rela-
tive abundance of enveloped particles and the enrichment of
highly infectious particles with the highest infectivity-HCV
RNA ratio was verified in each of the gradients used for the
analysis shown in Fig. 6C (see Fig. S5 posted at http://www
ing the notion that enveloped particles are enriched in samples
with higher specific infectivity. Furthermore, since the frac-
tions enriched in enveloped particles contained high envelope
(E2)/core ratios (see Fig. S6 posted at http://www.scripps.edu
/wieland/data/Gastaminza/FigureS6.tif), it is likely that their
characteristic bilayer corresponds to the viral envelope.
Since infectious HCV particles display heterogeneous buoy-
ant densities (Fig. 6A) and diameters (Fig. 5), we examined
whether the diameters of the enveloped particles were corre-
lated with their buoyant densities. We measured the diameters
of enveloped particles with different densities and observed
small, although statistically significant, differences in the diam-
eters of high-density (57.6 ? 10.1 nm, n ? 225) and interme-
diate-density (63.6 ? 4.5 nm, n ? 260) particles (P ? 2.10?13)
(see Fig. S7A posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS7.tif). These differences in diameters re-
flected a differential size distribution in which there was an
inverse relationship between particle diameter and buoyant
density (see Fig. S7B posted at http://www.scripps.edu/wieland
/data/Gastaminza/FigureS7.tif). Indeed, internal capsid-like
structures could be visualized in enveloped particles of signif-
icantly different diameters in our preparations (see Fig. S7C
posted athttp://www.scripps.edu/wieland/data/Gastaminza
The development of a cell culture system for HCV and its
optimization permitted production of high-titer virus stocks.
Purification of secreted particles by sequential ultracentrifuga-
tion yielded virus preparations containing most of the infec-
tious and noninfectious HCV RNA-containing particles.
Negative-stain EM and cryoEM analysis revealed a hetero-
geneous particle population with a mean diameter of ?55 nm
(Fig. 2), similar to patient-derived virus analyzed by rate-zonal
ultracentrifugation (51) and negative-stain EM (33, 37, 42, 57,
64). Detergent treatment resulted in total loss of infectivity and
a measurable reduction in the average virus particle diameter
from ?56 to ?45 nm (Fig. 3), again similar to detergent-
treated, patient-derived particles (51).
cryoEM analysis revealed an electron-dense bilayer around
some particles (Fig. 4). This feature allowed us to distinguish
two morphologically distinct particle classes (enveloped and
nonenveloped) that had different diameters (?60 versus ?45
nm, respectively), were approximately equal in frequency, and
accounted for over 90% of the total particle population (Fig.
5). Previous EM studies of patient sera described particles with
similar diameters (64), indicating that the cell culture-derived
virus recapitulates the morphologies of wild-type virus from
infected patients.
The ?60-nm E-type particles display a 5- to 6-nm thick
surface bilayer (Fig. 4) that most likely represents a lipid mem-
brane (25) and is consistent with removal of the envelope and
reduction in particle diameter with detergent treatment (Fig.
3B). The E particles were present in all of the infectious sam-
FIG. 3. Particles treated with 0.2% NP-40 are noninfectious and
display a decrease in diameter. Detergent-treated and untreated par-
ticles were subjected to ultracentrifugation in a continuous 10 to 50%
sucrose gradient. (A) Detergent treatment eliminated infectivity and
resulted in a substantial loss of HCV RNA. (B) Particle diameter
decreased from ?60 to ?45 nm with detergent treatment. Inset shows
representative negatively stained particles. Scale bar, 50 nm.
Page 7
ples we analyzed, and they were the dominant population in
preparations that displayed the highest specific infectivity (i.e.,
infectivity-RNA ratios) and E2-core protein ratio (Fig. 6; see
also Fig. S6 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS6.tif). Collectively, our results suggest
that infectious HCV particles are likely present within the E
particle population in these preparations. This is reinforced by
the fact that the average diameter of these D183 particles is
similar to the diameter of infectious JFH-1 virus particles (65
to 70 nm), which we previously calculated from their sedimen-
tation coefficient (20). In addition, it is compatible with the
diameter of infectious HCV estimated by size-based filtration
and inoculation into chimpanzees (8, 22).
The second major particle population (nonenveloped parti-
cles) is devoid of an observable bilayer (Fig. 5) and displays the
average diameter of detergent-treated particles (?45 nm), as
determined by EM (Fig. 3) and rate-zonal ultracentrifugation
(51). Unfortunately, detergent treatment reduced the number
of particles observable by cryoEM so that it was not possible to
compare their morphologies to those of untreated NE parti-
cles. In contrast to the E particles, which are relatively more
abundant in the highly infectious intermediate-density frac-
tions (Fig. 6), the NE particles are relatively more abundant in
high-density fractions that contain most of the viral RNA but
display relatively low infectivity (Fig. 6). Collectively, these
results suggest that the NE particles contain HCV RNA but
are unlikely to be infectious.
The multivesicular (MV) particles and large vesicles are
infection related since they are not found in control samples,
but they barely contribute to total HCV infectivity. Interest-
ingly, they display a striking resemblance to human exosomes
observed by cryoEM (56). Consistent with this hypothesis,
HCV structural proteins are associated with circulating exo-
somes in HCV-infected patients (45). Although MV particles
display very low infectivity in cell culture (Fig. 6), they could
conceivably play a role in natural infections. Additional exper-
iments aiming to determine the nature, biochemical composi-
tion, and functional properties of these exosome-like struc-
tures will be required to evaluate their functional relevance
during natural HCV infection.
The results presented in this study suggest that the bilayer
that differentiates E from NE particles constitutes the viral
FIG. 4. cryoEM showed that purified HCV particles are predominantly spherical and consist of subpopulations with different diameters. Note
that the large-diameter particles are surrounded by an envelope (arrowheads), which is not detectable in the small-diameter particles. Scale bar,
100 nm.
VOL. 84, 2010 cryoEM ULTRASTRUCTURE OF HCV PARTICLES11005
Page 8
envelope. First, the E particles are found predominantly in
preparations containing the highest E2-core protein ratio
(see Fig. S6 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS6.tif). Second, treatment of purified HCV
particles with detergent, which causes loss of viral envelope
and infectivity, results in a diameter shift from 60 to 45 nm
(Fig. 3), coinciding with the diameters of the E and NE parti-
cles, respectively (Fig. 5). If this is the case, infectious HCV
particles appear to contain an ?45-nm nonicosahedral capsid
that is not tightly associated with the viral envelope. These
features are clearly different from those of other members of
the family. In other members of the Flaviviridae family, the
viral envelope is usually not as distinct by cryoEM, where the
lipid bilayer is tightly associated with the capsid and appears
buried under a thick, smooth glycoprotein layer (34, 35). The
heterogeneous enveloped population with various diameters
FIG. 5. Purified virus preparations contained two major (enveloped and nonenveloped) and two minor (large vesicle and multivesicular)
subpopulations. (A) Gallery of representative electron cryomicrographs showing the four particle classes. Scale bar, 100 nm. (B) Histogram and
table of 2,086 particles from four independent virus preparations revealed roughly similar numbers of enveloped and nonenveloped particles, with
minor populations of large vesicles having a uniform interior density, and a few multivesicular particles that are probably nonviral and may
represent exosomes.
Page 9
surrounding an internal capsid resembles porcine reproductive
and respiratory virus ([PRRSV] Arteriviridae) particles (60).
Given the similarity in the sizes of the internal capsid and the
population of nonenveloped particles, it is possible that the NE
particles are capsids lacking an envelope. In fact, NE particles
accumulate in high-density fractions where core antigen levels
are the highest and the E2-core ratio is the lowest (see Fig.
S6 posted at http://www.scripps.edu/wieland/data/Gastaminza
It is unlikely, but formally possible, that the NE particles
display a viral envelope tightly associated with the capsid and
not visible in individual images. Indeed, a recent three-dimen-
sional reconstruction of noninfectious HCV virus-like particles
produced in insect cells suggested that ?50-nm particles have
an envelope tightly associated with the capsid (68). In that
study, these particles were compared with JFH-1 particles pu-
rified from isopycnic gradient fractions that contain the peak of
HCV RNA, have a diameter of 50 nm, and are devoid of any
evident bilayer (similar to particles in our high-density frac-
tions) (Fig. 6). Unfortunately, the morphological heterogene-
ity, relatively low particle yield, and lack of measurable sym-
metry of both E and NE particles precluded the image analysis
and reconstruction necessary to determine if the 45-nm NE
particles display a lipid bilayer.
We are grateful to Takaji Wakita (National Institute of Infectious
Diseases, Tokyo, Japan) for kindly providing the infectious JFH-1
molecular clone, Dennis Burton (The Scripps Research Institute, La
Jolla, CA) for providing the recombinant human anti-E2 and anti-HIV
IgGs, and Michael Houghton (Chiron) for providing the MS3 anti-
serum against HCV core. We thank Marlene Dreux, Urtzi Garaigorta,
Ken Takahashi, Stefan Wieland, and Barbie Ganser-Pornillos for their
expert advice and useful discussions. We are grateful to Erick Giang,
Christina Whitten, and Josan Chung for excellent technical assistance.
This work was supported by NIH grants R01-CA108304 (F.V.C.),
R01-AI079043 (F.V.C.), R01-A79031 (M.L.), and R01-GM066087
This is manuscript number 20607 from the Scripps Research Insti-
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62. Suloway, C., J. Pulokas, D. Fellmann, A. Cheng, F. Guerra, J. Quispe, S.
Stagg, C. S. Potter, and B. Carragher. 2005. Automated molecular micros-
copy: the new Leginon system. J. Struct. Biol. 151:41–60.
63. Tellinghuisen, T. L., K. L. Foss, and J. Treadaway. 2008. Regulation of
hepatitis C virion production via phosphorylation of the NS5A protein. PLoS
Pathog. 4:e1000032.
64. Trestard, A., Y. Bacq, L. Buzelay, F. Dubois, F. Barin, A. Goudeau, and P.
Roingeard. 1998. Ultrastructural and physicochemical characterization of
the hepatitis C virus recovered from the serum of an agammaglobulinemic
patient. Arch. Virol. 143:2241–2245.
65. Wakita, T., T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K.
Murthy, A. Habermann, H. G. Krausslich, M. Mizokami, R. Bartenschlager,
and T. J. Liang. 2005. Production of infectious hepatitis C virus in tissue
culture from a cloned viral genome. Nat. Med. 11:791–796.
66. Yeager, M., J. A. Berriman, T. S. Baker, and A. R. Bellamy. 1994. Three-
dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron
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67. Yi, M., Y. Ma, J. Yates, and S. M. Lemon. 2009. Trans-complementation of
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VOL. 84, 2010cryoEM ULTRASTRUCTURE OF HCV PARTICLES11009

subset: commoncrawl


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Head to the shores of Lake Erie this spring and save 50% at the Hyatt Regency Buffalo, which "soars both into the sky and above its competition as the most elegant of the downtown chains" (Frommer's).
Shopping, casinos, wining and dining await on the U.S. side of Niagara with a $79 overnight package that saves more than 65% on regular rates through June.
An award winning, Georgian-style brick mansion with "a hushed regal air to it" (Fodor's) is offering a Travelzoo-exclusive getaway for two, including select weekends, saving over 50% on regular rates.


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England unveil latest hi-tech kit
ESPNscrum Staff
August 1, 2011
England's kit for this year's Rugby World Cup © Getty Images
England have officially unveiled their new playing kit and the controversial all-black change strip.
The new white kit will be worn for the first time when England kick off their Rugby World Cup warm-up clashes against Wales in Cardiff on August 13 and again against Ireland at the Aviva Stadium in Dublin on August 27. The new change kit will be worn for the first time when England play Wales at Twickenham this Saturday and again against Argentina in their World Cup showdown in Dunedin on September 10 due to a clash with the light blue and white of the Pumas.
The shirts, that will retail at £90 for the authentic version and £50 for the replica, have been produced by long-term partner Nike who claim every element of the playing kit has been "tailored and improved to reflect player feedback" from past seasons to make this the "most complete England Rugby kit to date".
England's Chris Ashton, who scored a magnificent try when England beat Australia in their grey change kit last November, laughed off the controversy surrounding their latest change strip. "You are more worried about playing for your country than what colour kit you are wearing. If we played in pyjamas it wouldn't bother me," said the Northampton wing. "The grey one worked well - we played well in it - let's hope it is the same with the black one."
According to the manufacturers, "The tight-fitting playing shirt is made of Nike Dri FIT fabric - a technology engineered to provide sweat management for athletes, pulling moisture away from the body to optimize player temperature and help them keep them cool and dry. All materials are strength-tested at Loughborough University to ensure they remain strong but flexible on game day to prevent tearing.
"Based on player feedback, the collar has elastic within the neckline for the first time, keeping the neckline as close to the body as possible to help prevent shirt pulling whilst providing comfort. There is also a silicone gripper at the hemline of the playing shirt to help keep it tucked in and close to the body. The silicone hem features the word England in its design.
"The clean white home shirt design features a subtle matt/gloss white check pattern on the front panel of the shirt whilst the change shirt has a matt/gloss black check in the same area.
"The gripper panels on the shirt, to give players more control of the ball are now across the shoulders, pectoral area and stomach area of the shirt and arranged in a check pattern. The iconic rose emblem is made from Nike Considered (recycled) yarn and heat-applied to the shirt to avoid puncturing and weakening the fabric.
"For the first time the shorts feature a new flock hand dry print to help players keep hands dry during games. And the socks, which feature support material for the first time, have a no-contact toe seam and enhanced fit footbed to reduce blisters and improve comfort."
© ESPN Sports Media Ltd.
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Huw Richards assesses where Wales are after a mixed Six Nations, with front row seats still very much available for the World Cup
Attacking revolution will be fleeting
John Mitchell lapped up the action on 'Sensational Saturday' - but warns not to expect a repeat come Rugby World Cup time later this year
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Craig Dowd warns England, Ireland and Wales they should play to their strengths rather than those of the All Blacks and the Wallabies
Six Nations prospects for the World Cup
Tom Hamilton runs the rule over just where the six countries stand ahead of the global gathering in September


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WIMBLEDON, England -- Venus Williams still hopes her star is rising while Lindsay Davenport sees the twilight. On Tuesday at Wimbledon, Williams and Davenport met for the 22nd time -- both former champions seeking a win to boost their battered confidence.
In the end, it was Venus who came away with the 6-2, 2-6, 6-2 victory, speaking of future titles, while Davenport spoke of playing her last Wimbledon singles match.
"It definitely ran through my mind when I was out there, that that could be my last singles match out there," Davenport said. "But you know, I don't want to blow it out of proportion. Absolutely no decision by far has been made."
Davenport knows her opportunities for major wins are dwindling.
"Time's running out," she said. "I don't know how many more I'll be playing in to know that. & It just wears on you after a while. I'm 27. I want to feel like I can keep getting better, and I want to feel like I'm one of the top players. I don't want to be, you know, six or whatever the ranking is, through 20. That's just where my heart is."
If it was to be her last match here, it didn't go the way she would have wished. In the first game, Williams showed she was ready for a fight as she won every point and finished with an ace. As she took a 2-0 lead, she had no unforced errors, a far cry from the woman with 75 unforced errors in her fourth-round match at the French Open.
"Even the first service game," Davenport said, "I made something like nine out of 10 first serves and still got broken. I don't think that would happen against a lot of players."
After losing the first set, Davenport didn't hang her head -- as she often does. She fought back to even the match despite letting a 40-0 lead slip away at 1-0 in the second set. But just as Davenport stepped up the level of competition, so did Williams, running away with the third set.
Williams will play Belgian Kim Clijsters in the semifinals on Thursday. It gives Williams an opportunity to help her sister keep her No. 1 ranking. Williams leads their series 4-2; they've split their past two matches, although Williams says she doesn't count the walkover at the WTA Championships. In fact they've never fought an all-out battle.
"I don't think we've played each other where we're both at our best, I think," Clijsters said after defeating Silvia Farina Elia 5-7, 6-0, 6-1.
Williams has struggled to find her best lately. She's chalked up only one tournament win this year -- against Clijsters -- and has lost four of the past five Grand Slam tournament finals to her sister. She's played a limited schedule, her desire to play the game has been questioned and her rank has slipped to No. 4.
"I think that if I'm listening to what everyone says, of course, it's going to be hard," Williams said. "But I have to listen to what I think, and I think that I had a pretty good year. And it's impossible to be a shining star every time. If so, then I guess it wouldn't be as much fun for the fans.
"What I'm saying, it's impossible to be in the winner's circle every time. You don't learn as much if you don't lose a few times."
And has she learned anything from her sister?
"I think, for sure, Serena just always had the better shot last year, for no matter who she was playing," Venus said. "She always had the right answer. She definitely was very motivated to do well and to win, and that's really what it takes."
What motivates Venus Williams is being a champion again.
"I don't come to the tournaments just to have a good showing," she said. "And if I don't win a tournament, I don't get extremely upset. But I would love to win, just like all the other competitors who are going to be in the semifinals. And I would just have to play to win."
Cynthia Faulkner is the tennis editor for ESPN.com.


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How Long Do I Wait To Get Tested After Blood In Food
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
What Is HIV?...you cannot get the health care and treatment you need if you do not know your HIV status. Being unaware of your status also means that you could pass HIV to others without knowing it. For women who plan to become pregnant, testing is especially important. If a woman is infected with HIV, medical care and certain drugs given during pregnancy can lower the chance of passing HIV to her baby. If you test HIV-negative you can take steps to stay...
Ask the Experts
Will Donate! Blood in Refrigerated FoodI just ate a wrap that I think had blood on it and was refrigerated (which I'm worried preserved the HIV). After eating the first half I noticed a small blood smear on the second half of the wrap along the cut line. I am really worried. This...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...Hi,Relax Max. Your HIV-acquisition risk is nonexistent. HIV doesn't survive very long at all outside the body, even if kept in the refrigerator! It's a virus -- frevinsakes! -- not a banana cream pie!Dr. Bob Read more »
The accuracy of OraQuick/Orasure? Cant even mini woo hoo?? (RELIABILITY OF ORAQUICK ORAL TESTS, 2009)Hi Dr Bob,I just did a rapid OraQuick or Orasure, not sure. The doc swabbed my lower and upper gums, not sure if they swabbed my cheeks, but he swabbed it very quick (I am sure not more than 1-2min) and I am not sure if it more than 1 min and I have...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...continues to meet Food and Drug Administration (FDA) requirements. Overall, oral fluid rapid tests have performed well and make HIV testing possible in many venues where performing a blood draw or finger stick is impractical or impossible. The... Read more »
Oraquick Advance Test Confusion (RELIABILITY OF ORAQUICK TESTS, 2009)Dr BobI tested negative using the Oraquick Advance HIV 1/2 rapid test. I started reading some things on line that stated the accuracy of the rapid test was not as good as the standard ELISA test and had a higher rate of false negative test. This...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...question!1)With all the recent reports on Orasure testing, would you say the mouth swab and finger prick methods are still accurate testing methods as long as you test after the window period?2)Do you recommend using those methods?Thanks Dr. Bob... Read more »
Do HIV carrirers test negative (HIV CARRIERS) (HIV BASICS)DO HIV carriers test negative? (I did an HIV 1&2 test which came back negative. But someone told me that I could still be a carrier. The test was done six (6) months after my last possible exposure.
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...considering becoming pregnant and thinks she may have been exposed to HIV -- even if the exposure occurred years ago -- should seek testing and counseling. In the U.S., mother-to-infant transmission has dropped to just a few cases each year... Read more »
Should Gay Men Be Allowed to Donate Blood?...person who has had tuberculosis must wait two years after successful curative therapy to give blood. The list of restrictions is long and complex and not all seem obviously justified. (See box: The Top 52 Reasons for Not Being Able to Donate... Read more »
Mycobacterium Avium Complex (MAC)...maintenance therapy, azithromycin plus ethambutol are the preferred drugs. The Bottom Line Researchers are developing and testing new drugs and drug combinations that are more effective and have fewer side effects. More novel treatment... Read more »
Black America and HIV Testing...standard antibody tests are highly accurate, with only a remote risk of a false positive result. Standard antibody tests have long been common in a variety of venues, including physicians' offices and stand-alone HIV testing and STD clinics. An... Read more »
Blood Work: A Complete Guide for Monitoring HIV...to have the same type of test (bDNA, PCR, etc.) done each time. If your doctor sends you to the same place to give blood for testing, it's likely the same labs and types of tests are being used. If you move or if you change doctors or health... Read more »
Connect With Others
Chances that I am HIV positive?
Posted by Tallscot, 1 Reply...2008. I am worrying myself sick and can hardly eat and very nervous and paranoid that i have HIV now!! ;o( And dreading the long wait till monday night and when the doctor tells me the HIV blood result! Please reply back and let me know with my... Read more »
I had hiv symptoms for the last two years and still..............
Posted by testednegative...remedy. I would take special care to reveal to him only one symptom as i feared that he would suspect me and send me to hiv testing. I blamed myself for all this and felt extremely depressed. Ooh what days were those, i would think about all... Read more »
just tested negative
Posted by Anonymous, 1 Reply...gathered up the courage to get tested...i have not exactly lived a sin free life so i was scared to death...after waiting 8 long days...with no sleep and lots of crying i went to pick up my test results today...my thinking was better to know what... Read more »
Posted by shadow1, 3 Repliesarticle in the LA Times about new tests being considered by the FDA. http://www.latimes.com/news/local/la-me-rapid28sep28.story?null Read more »
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Porsche Customizer Puts a 1972 Kharmann Ghia Body on a Boxster S
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Porsche Customizer Puts a 1972 Kharmann Ghia Body on a Boxster S
SEMA wrapped last week and we're still sorting through all the pictures that came our way from friends, readers and sponsors. One of the more interesting shots came from frequent contributor Roger Garbow. The picture below was taken outside the Las Vegas Convention Center and shows what appears to be a spectacularly restored and updated 1972 Kharmann Ghia. However, after a closer inspection it's apparent that this Kharmann Ghia is more than it seems.
Porsche restorer, customizer and mechanic Tony Miller of
in York, Pennsylvania was commission by a customer to create this one off Porsche-Kharmann compilation. Using a 2001 Boxster S for the chassis and drive-train, Miller widened and customized the body of a '72 Kharmann Ghia to fit the Boxster's frame. Almost every body piece was replaced during the process. According to Miller, "
the only exterior sheet metal we retained from the Boxster was the tonneau cover in the rear and the rocker panels which are integrated in the rear quarter panels and Ghia fenders.
While the drive train of the Boxster S remains mostly stock, Miller did swap out the exhaust for one from Fabspeed and the suspension was upgraded with H&R springs. When we asked about how the change in body work and aerodynamics affected the driveability we were surprised to find out they didn't yet know. The car looks so good in the picture above we just assumed it was running. However, Tony tells us it's still a rolling chassis at this point as there is still some mechanical work needed to get the Boxster radiators and condensers modified to fit in the shorter Kharmann Ghia body. He expects things to be completed by the end of the year and we should have an update for you then.
There's not yet a ton of additional information on this customized Boxster/Kharmann Ghia on Tony's site, but there are a
few more pictures
contact details
if you are interested in doing something similar to this yourself. Let us know if you do, we want to write about it!
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The writer, fashionista, model, an DJ talks sneakers, self esteem, and racism.
The Brooklynite poet and lyricist Aja Monet drops her EP "Courage".
Chang waxes poetic about how to hustle, her hair game, and being fit.
Sebastian Mikael sits down and talks Sweden, Speechless, and one night stands.
The British singer talks reincarnation, grime, and Lemony Snicket.
Stella Blu talks travel, feminism, and Prince.
Mal and Tallulah give us some pre-college words of wisdom.
The 18-year-old artist talks Photoshop, technicality, and the MoMA.


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Rate Dr. Robert J. Zehr
Dr. Robert J. Zehr's Ratings
Dr. Zehr has replaced 2 knees for me -- the first 8 years ago, and the second 7 weeks ago. He is a first-class surgeon, and his staff is extremely knowledgeable and helpful. The booklet he gives you telling what to expect before, during, and after surgery is an invaluable resource. In my pre-op physical it was recommended I get a heart scan, and Dr. Zehr's office got me an immediate appointment so the knee surgery could continue as scheduled. Any questions I had were answered promptly and I felt cared for and valued. Although there are many joint replacement surgeons in Naples, I feel privileged that I chose Dr. Zehr and his team to take care of me, and I would totally recommend them. Because I had an outstanding surgeon and an excellent physical therapist (Mark Johnson of Naples Physical Therapy) I am well on my way to a full recovery.
Was this rating useful? 0 [[ userState['ratings'][2756061].votes_count ]]
Dr. Zehr is top notched with this hip surgery. The procedure was done in a day, and I was home the following day. Take his recommendation, if the hip need replaced, do it. Don't procrastinate. The quicker you do it, the quicker you return to a less pain filled life. The whole secret is to follow his directions, get off of the pain medication and do physical therapy plus WALK.
Was this rating useful? 0 [[ userState['ratings'][2745191].votes_count ]]
Dr. Robert J. Zehr's response
How right you are - you must do the work of physical therapy. It's not easy but it's critical. Congratulations on your progress.
Submitted March 11, 2015
The only addition I would add to his WONDERFUL guide book is the caution to raise your legs above the heart post surgically to reduce swelling. Once I did that, everything got easier. Thanks for a great service beginning to end!
Was this rating useful? 0 [[ userState['ratings'][2738481].votes_count ]]
Dr. Robert J. Zehr's response
Good advice! We're so glad you had a successful outcome.
Submitted March 11, 2015
I cant say enough about how GREAT the staff, facilities, processes and Doctor Zehr all worked together to replace both of my hips over a 3 month period. Everyone at Zehr was helpful, the facility was great looking and spotless, and they led me thru the whole process with an instruction binder and answering questions to the fullest.
They never balked at helping me in any way possible.
I interviewed 4 DRs and I chose Dr Zehr and his staff. I am very pleased i did.
If you need Ortho treatment....this is place for results!!!!!
Was this rating useful? 0 [[ userState['ratings'][2705342].votes_count ]]
Dr. Robert J. Zehr's response
We are pleased too! Thank you for your insight, we are glad to know the pre-surgery education was appreciated!
Submitted Feb. 23, 2015
On a scale of one to ten, I'd give Dr. Zehr and his staff an 11. As a result of their skill, professionalism and care, I am pain-free and able to be fully active again.
Was this rating useful? 1 [[ userState['ratings'][2684875].votes_count ]]
Dr. Robert J. Zehr's response
We are so glad to hear that! (And we love your math!)
Submitted Feb. 23, 2015
If my left hip ever needs to be replaced I would absolutely go to Doctor Zehr. He and his staff make it almost easy to correct what was a very painful right hip.
Was this rating useful? 1 [[ userState['ratings'][2678074].votes_count ]]
Dr. Robert J. Zehr's response
And if your left hip ever needs replacing we would be honored to correct that for you as well, "because living with pain isn't really living!"
Submitted Feb. 23, 2015
Right hip replacement - results have been outstanding.
Was this rating useful? 1 [[ userState['ratings'][2664213].votes_count ]]
Dr. Robert J. Zehr's response
We are very glad to hear that you have had outstanding results - that means you did the hard work of rehab!
Submitted Feb. 23, 2015
Very professional. Very knowledgeable. I am very satisfied.
Was this rating useful? 1 [[ userState['ratings'][2663737].votes_count ]]
Dr. Robert J. Zehr's response
Thank you for letting us know you are satisfied with the results of your surgery, we do strive to keep up with the latest and greatest orthopaedic advancements.
Submitted Feb. 23, 2015
I am 59yrs old and developed osteoarthritis in my hips due to sports, work and very active life style. Jan 2011 I had my left hip replace in Ohio by a top orthopedic surgeon. At that time the minimally invasive posterior approach was being used. June of 2013 we moved full time to Ft. Myers Fl. Needing my right hip replaced I went about researching the minimally invasive anterior approach that has been gaining in popularity since my first THR in 2011. This is what brought me to Zehr Center for Orthopedics and Dr. Zehr.
I won't go on with what a lot have said because I found Dr Zehr and staff to be the best you can be also. Although I do have a unique opportunity to compare the 2 approaches by 2 different top notch surgeons.
I was skeptical about the claims being made about this different anterior approach because I did have a rough time with my first THR and after all you are replacing a hip, by no means minor surgery. In the end it all far surpassed my expectations. After surgery I was up in 4hrs with a walker and standing full weight on both legs talking to nurses at the nurse station in the middle of the floor. I spent a total of 28hrs in the hospital and I was using a cane when it was time to go. I could have walked out with the cane but hospital policy is wheelchair. I had my surgery on Nov 25th went off the cane 6 days after surgery and walked in unassisted to my follow up appointment Dec 4. It took 1 month with my left THR to get off the crutches or walker and months before I stopped using a cane.
Truly I don't know how it could have gone better. Yes it hurt and still does but the pain is different and easily managed. My leg strength is 10 times better than it was with previous THR. There is a vast difference in what muscles are damaged and time needed to get back to normal.
I could go on but will end this by saying Dr Zehr and staff are in a class of there own. The minimally invasive anterior approach lives up to and surpasses the hype and in my case I look to be back to normal activity in a month. There will still be pain and there will still be reduced flexibility, but there really is no comparison. At times sitting now I forget it was just done.
The biggest key to success with joint replacement beyond the surgeon is support at home. My wife has cried a couple of times with relief at the difference this time and our lives getting back to normal so easily and quickly. Thanks to Dr. Zehyr!
Was this rating useful? 1 [[ userState['ratings'][2643740].votes_count ]]
Dr. Robert J. Zehr's response
We love to hear good, honest testimony - and are glad you are recovering so well.
Submitted Feb. 23, 2015
I broke my hip when I was 18. It healed but that was 23 years ago. Over the last 10 years the pain in my hip has gotten to the point of unbearable. By last fall I couldn't walk 30 feet without extreme pain and the use of a cane. I had been to 2 other doctors who both said I was too young and too overweight for hip surgery, it's hard not to be over weight when you can't walk.
I read an article about anterior approach hip surgery and then started to look for a doctor who had a lot of experience doing the procedure. I found Dr Zehr and was surprised how quickly I was able to get an appointment. I was very comfortable after meeting him and he scheduled surgery for several weeks later.
The hospital was very nice and my surgery and recovery went very well. The pain was less then I expected, actually there was little to no pain in my hip, the majority of the pain was caused by the nerves and muscles that get moved around during the operation, but with lots of ice bags and some pain medication, it was really no big deal. I wouldn't say that I'd look forward to getting another hip replacement but if I did have to, I wouldn't dread it like the first time. There is no other doctor that I would go to other then Dr Zehr and there is no other hospital that I would go to then Physicians Regional in Naples.
It's been 10 months and I am a brand new person. This past summer, only 4 months after my surgery, I started traveling again and went almost every state and most of Canada. My friends can't believe the difference and now I walk faster then everyone. If you need a hip replacement, go to Dr Zehr and get it done now.
Was this rating useful? 1 [[ userState['ratings'][2643712].votes_count ]]
Dr. Robert J. Zehr's response
Great advice! Thank you for sharing about the difference direct anterior approach hip replacement surgery has made in your life.
Submitted Feb. 23, 2015
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Whether youre a fan of the classics or contemporary nonfiction, As Seen on TVs DVD collection has you covered from Auntie Mame to a bevy of History Channels docu-series. Shop DVD sets for great TV programming!
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Back Street: Two Film Collection DVD
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Turner Classic Movies and Universal Studios Home Entertainment present Back Street: Two Movie Collection. BACK STREET (1941) The 1941 version of Back Street, directed by Robert Stevenson, stars Margaret Sullavan and Charles Boyer with vivid supporting roles by Richard Carlson, Frank…
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The last desperate fight that changed the course of history. Five months after D-Day, most American soldiers think the German army is broken. The Germans think otherwise. In an attempt to buy time to fill the skies with their invincible new jets, they launch one fast, furious offensive: the…
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Experience the visual splendor, thundering action and towering drama of this record-setting winner of 11 Academy Awards(R) including Best Picture. Charlton Heston brings a muscular physical and moral presence to his Best Actor Oscar(R)-winning role of Judah Ben-Hur, a Jewish nobleman in…
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This two-disc set offers the best so far from this popular A&E series. The Robertsons have taken their Louisiana mom-and-pop operation,Duck Commander, and turned it into a sporting empire by making top-of-the-line duck calls and decoys out of salvaged swamp wood. Millions enjoy watching…
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Best Of Duck Dynasty: In The Blind DVD
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Includes the following episodes from Season 1: Sauvignon Beard Plan Bee Daddy's Got A Gun Fishin' For Business Redneck Roadtrip Winner, Winer Turkey Dinner Willie Stay Or Willie Go
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Best Of Sherlock Holmes Collection DVD
3.5(2 reviews)
The best of Sherlock Holmes on four DVDs! This super-sleuth collection features nine of the very best Sherlock Holmes films starring Basil Rathbone, Arthur Wontner, Raymond Massey, and more plus 20 episodes of the critically acclaimed series starring Ronald Howard, hailed as the most…
- $39.98
Bogie & Bacall: The Signature Collection DVD
4.9(24 reviews)
They met on the WB lot. The year was 1944. "I just saw your screen test," Bogart said to Bacall. "I think we're going to have a lot of fun together." And so it began... Listed as the Greatest Male Star of All Time and one the Greatest Female Legends by the American Film Institute, Humphrey…
- $19.98
0.0(No reviews)
Following the landmark success of the acclaimed HBO® series Russell Simmons Presents Brave New Voices, this all-new, one-hour HBO special presentation from executive producers Russell Simmons and Stan Lathan celebrates youthful excitement, creative expression, and inspired stage…
- $14.98
Cabaret: 40th Anniversary Special Edition DVD
4.0(6 reviews)
Liza Minnelli stars in this musical extravaganza set in a Berlin cabaret in 1931, where the rising tide of Nazism has direct effectson the lives of several individuals. A female girlie club entertainer in Weimar Republic era Berlin romances two men while the Nazi Party rises to power around them.
- $49.98
Cary Grant: The Signature Collection DVD
0.0(No reviews)
Destination Tokyo What the World War II - era film Air Force is to the skies, Destination Tokyo is to the seas. In the only military-action film he made during the war, Cary Grant plays Captain Cassidy of the U.S. submarine Copperfin, skippering his torpedo-laden thunderfish with…
- $64.98
Casablanca: 70th Anniversary Edition (Blu-ray + DVD)
4.0(1 review)
Casablanca: easy to enter, but much harder to leave, especially if you're wanted by the Nazis. This is how the story goes on the Casablanca: 70th Anniversary Edition DVD and Blu-ray. Victor Laszlo (Paul Henreid) is a Resistance leader, whose only hope is Rick Blaine (Humphrey Bogart), a…
- $34.98
Centennial: The Complete Series (Repackage) DVD
5.0(2 reviews)
It is their story. It is our story. It is the story of America.Journey to the Wild West in the sweeping, sensational epic drama Centennial: The Complete Series! Relive the grand hopes, dreams, loves, and adventures of generations of residents in Centennial, Colorado - from their risky…
- $19.98
Christianity: The First Two Thousand Years DVD
4.0(4 reviews)
From the Crucifixion to the Crusades, the Reformation to the sweeping changes of Vatican II, CHRISTIANITY: THE FIRST TWO THOUSAND YEARS takes a comprehensive look at the events and people who have influenced and been a part of the epic journey that is the evolution of the faith. Drawing on…
- $59.98
Clark Gable: The Signature Collection DVD
4.3(3 reviews)
Named as the seventh greatest actor on AFI's List of "50 Greatest Screen Legends," Clark Gable reigned supreme as a screen icon during the 1930's and 40's. Six of Gables 75 feature films are remastered and now available on DVD in the new Clark Gable: The Signature Collection. Boom Town…
- $14.98
Classic Westerns: 10 Movie Collection DVD
4.7(3 reviews)
Films Include: When The Daltons Rode When young lawyer Tod Jackson (Scott) arrives in pioneer-era Kansas, he learns his rancher friends, Grat and Bob Dalton (Donlevy and Crawford), are in danger of losing their land in a crooked development deal. Jackson tries to help them, but a fake…
- $49.99
Columbia Pictures Film Noir Classics IV DVD
3.8(18 reviews)
Sony Pictures Home Entertainment, The Film Foundation and Turner Classic Movies again partner to present the fourth collection in this series, Columbia Pictures Film Noir Classics IV. These five films, all fully restored and remastered and never before released on DVD, showcase the work of…
- $19.99
Desert Storm: The Ultimate War DVD
0.0(No reviews)
The men who ran the show provide the definitive insider's account of a dominating victory that was the spectacular payback of a winner-take-all gamble.
- $279.98
Dexter: The Complete Series Blu-ray
0.0(No reviews)
Dexter is a crime drama about Dexter Morgan, a man who leads a double life as an incredibly likeable forensics expert for the Miami Police Department and as an emotionless vigilante serial killer. Taught by his foster father to harness his lust for blood and killing, Dexter lives by his own…
- $179.98
Dexter: The Complete Series DVD
5.0(4 reviews)
Dexter is a crime drama about Dexter Morgan, a man who leads a double life as an incredibly likeable forensics expert for the Miami Police Department and as an emotionless vigilante serial killer. Taught by his foster father to harness his lust for blood and killing, Dexter lives by his own…
- $29.98
4.8(4 reviews)
Dexter is a crime drama about Dexter Morgan, a man who leads a double life as an incredibly likeable forensics expert for the Miami Police Department and as an emotionless vigilante serial killer. Taught by his foster father to harness his lust for blood and killing, Dexter lives by his own…


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Article — From the October 1967 issue
Letter from academe
The life of Professor Riley
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Article — From the October 1967 issue
The life of Professor Riley
Article — From the February 1967 issue
Article — From the July 1965 issue
Article — From the March 1964 issue
Article — From the August 1960 issue
An eyewitness report on the churches in Eastern Europe
Article — From the December 1956 issue
Article — From the February 1954 issue
Their cause and cure
By Justin Smith
Acreage of a Christian nudist colony under development in Florida:
Continuing Care, Inc. (Venice, Fla.)
Florida's wildlife officials decided to remove the manatee, which has a mild taste that readily adapts to recipes for beef, from the state's endangered-species list.
A 64-year-old mother and her 44-year-old son were arrested for running a gang that stole more than $100,000 worth of toothbrushes from Publix, Walmart, Walgreens, and CVS stores in Florida.
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"He could be one of a million beach-bound, black-socked Florida retirees, not the man who, by some odd happenstance of life, possesses the brain of Albert Einstein — literally cut it out of the dead scientist's head."
© 2014 Harper's Magazine. April 2015 cover painting: Man Seated by Rockwell Kent


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My wife and I go to Ruby Tuesday mostly for the excellent salad bar. I have almost always ordered the creole catch, which is a nice whitefish (tilapia) with creole seasoning. The fish is always properly cooked, but the sides (broccoli or green beans) tend to be badly overcooked, even when the wait staff is given explicit instructions about this problem.
Fortunately, on our most recent visit, ordering the fish allowed you to have the salad bar and a baked potato as the two sides, thus avoiding the soggy vegetables. My wife, who has had mixed experience with various burgers and other dishes, finally followed my lead last time and had the fish. She was converted.
If you own or manage Ruby Tuesday, register now for free tools to enhance your listing, attract new reviews, and respond to reviewers.


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How Does It Grow? Lettuce
Give your child a life science lesson with this fun worksheet. As she cuts and pastes the pictures in the right places, she'll learn a little about the process lettuce goes through as it grows. Can she get everything in the right order?
Don't forget to check out the rest of the series here.
Topics: Second Grade, Life Science
Updated on Sep 20, 2012
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Saint Mazie has been selected to be a Book at Bedtime this summer on BBC Radio 4! Also the wonderful publisher of The Middlesteins, Schöffling & Co., will also be publishing Saint Mazie in 2016. And finally, check out Jami's latest essay in The New York Times Magazine: Track Changes.
Saint MazieBig-hearted, bawdy Mazie Phillips rules the Bowery during the Depression Era.
The MiddlesteinsA Midwestern family seeks hope and love even in the face of heartbreak and loss.
The Melting SeasonCatherine Madison is on the run with a suitcase full of cash.
The Kept ManJarvis Miller has been waiting for her husband to die for six years.
Instant Love: FictionThree young women explore love and loneliness across America.


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OP Senior Member
Thanks Meter: 16
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Join Date:Joined: Sep 2011
Also, is there a way to flash T-Mobile on here? if not thats not a big deal.. Thanks!!
Theres a thread in the general section on how to sim unlock all galaxy S3's and the galaxy note.
I would recommend selling your AT&T S3 and buying the tmobile S3... That way you don't have to put up with 2g (edge)... You might be able to get 3g if your in an area that has been reformed.
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Baseball Coaches
Michael Carroll Head Baseball Coach
Mike Carroll is in his 16th season as the women's head hockey coach at Gustavus. Within his tenure behind the Gustie bench, Carroll has turned Gustavus women's hockey into one of the most successful programs in NCAA Division… (continue reading)
Nate Johnson Assistant Coach Football and Baseball
Nate Johnson begins his third season on staff for the Gustavus Adolphus football team, serving as the team's defensive backs coach and video coordinator. Johnson will also begin his second season as an assistant baseball coach… (continue reading)
Matthew Lewis Assistant Baseball Coach
Matt Lewis enters his fourth season on the coaching staff of the Gustavus baseball team in 2014, serving as the team's pitching coach. Lewis joined the staff last season after a stellar pitching career at the NCAA Division III… (continue reading)


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Our new devotional "If God Loves Me, Why Did I Lose My Job?" explores the struggle of unemployment. Sign up here.
8:33 Get behind me, Satan. Satan is now at work even among Jesus' own disciples, not only in Judas but even in Peter, whose intervention would have annulled the plan of redemption and accomplished Satan's goal.