Stability of Hepatitis C Virus, HIV, and Hepatitis B
Virus Nucleic Acids in Plasma Samples after Long-Term
Storage at –20oC and –70oC
Cristina Baleriola1, , Harpreet Johal1, , Brendan Jacka1, Sandra Chaverot1, Scott
Bowden2, Sara Lacey2 and William Rawlinson1,3,4*
The storage of biological samples may affect detection of viral nucleic acid, yet the stability
of viral nucleic acid at standard laboratory storage temperatures (–70°C and –20°C) has not
been comprehensively assessed. Deterioration of viral RNA and DNA during storage may
affect the detection of viruses, thus leading to an increased likelihood of false-negative
results on diagnostic testing. The viral loads of 99 hepatitis C virus (HCV), 41 HIV, and
101 hepatitis B virus (HBV) patient samples were measured before and after storage at –
20°C and –70°C for up to 9.1 years using Versant branched DNA assays, Cobas Monitor
assays, and/or AmpliPrep/AmpliScreen assays. Clinical samples stored at –20°C for up to
1.2 years and at –70°C for up to 9 years showed a statistically significant difference from
baseline with respect to HCV RNA titer, although this difference was not greater than 0.5
log10 unit. The concentration of HIV RNA in clinical samples stored at –20°C for 2.3 years
and at –70°C for up to 9.1 years did not differ significantly from the baseline viral load.
HBV DNA-positive clinical samples stored at –20°C for up to 5 years and at –70°C for up
to 4 years differed significantly in viral load. In all studies, however, the loss of viral load of
HCV, HIV, or HBV in clinical samples tested after storage at –20°C and –70°C for up to 9
years ranged from 0.01 to 0.35 log10 IU/ml and did not exceed 0.5 log10, which is the
estimated intra-assay variation for molecular tests. Hence, the loss was considered of
minimal clinical impact and adequate for the detection of HCV, HIV-1, and HBV nucleic
acids using nucleic acid assays for the assessment of the infectious risk of cell, blood, and
tissue donors.
http://jcm.asm.org/cgi/content/short/49/9/3163
Development of a Multiplex Bead-Based Assay for
Detection of Hepatitis C Virus
Bruna P. F. Fonseca1,*, Christiane F. S. Marques1, Lílian D. Nascimento1, Marcelle B.
Mello1, Leila B. R. Silva1, Nara M. Rubim1, Leonardo Foti2,3, Edimilson D. Silva1,
Antonio G. P. Ferreira1 and Marco A. Krieger2,3
ABSTRACT
Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting
approximately 3% of the human population. Although HCV detection is currently based on
reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate,
and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5,
and Combined, we describe a new bead-based multiplex test capable of detecting HCV
infection in human serum samples. The first analysis, made in a singleplex format, showed
that each antigen coupled to an individual bead set presented high-level responses for antiHCV-positive reference serum pools and lower-level responses for the HCV-negative pools.
Our next approach was to determine the sensitivity and specificity of each antigen by
testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format,
the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and
55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%.
All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex
assay, which resulted in increased sensitivity and specificity values, performing with 100%
sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex
assay were 100%. Although preliminary, this 4-plex assay showed robust results that,
aligned with its small-sample-volume requirements and also its cost- and time-effectiveness,
make it a reasonable alternative to tests currently used for HCV screening of potentially
infected individuals.
http://cvi.asm.org/cgi/content/abstract/18/5/802
TaqMan Real-Time Reverse Transcription-PCR Assay
for Universal Detection and Quantification of Avian
Hepatitis E Virus from Clinical Samples in the Presence
of a Heterologous Internal Control RNA ,
Salome Troxler, Ana Marek, Irina Prokofieva, Ivana Bilic and Michael Hess*
Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In
this study, the development of the first duplex TaqMan real-time reverse transcription-PCR
(RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and
probes binding within relatively conserved open reading frame 3 (ORF3) were designed.
Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard
for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in
vitro and used as a heterologous internal control for both RNA isolation and real-time RTPCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression
squared value of 0.996, and a sensitivity of approximately 3.6 x 103 copies per reaction
mixture when in vitro-transcribed RNA was used as the template. The presence of in vitrotranscribed heterologous internal control RNA did not affect amplification of avian HEV
RNA compared to that achieved by the single assay. The sensitivity of the real-time RTPCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly
specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes
did not produce positive signals. All tested field samples with virus belonging to different
avian HEV genotypes were successfully detected with this new duplex TaqMan real-time
RT-PCR assay.
Ultrasensitive Quantification of Hepatitis B Virus
A1762T/G1764A Mutant by a SimpleProbe PCR Using a
Wild-Type-Selective PCR Blocker and a Primer-BlockerProbe Partial-Overlap Approach
Hui Nie1, Alison A. Evans2,3, W. Thomas London4, Timothy M. Block1,3,5 and
Xiangdong David Ren1,5,6*
Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core
promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of
liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses
may help in predicting the risk of HCC. However, the viral genome tends to have
nucleotide polymorphism, which makes it difficult to design hybridization-based assays
including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early
developmental stage is even more challenging, as the mutant is masked by excessive
amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory
PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the
amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the
proportion of the mutant could be increased by about 10,000-fold, making the mutant more
readily detectable by downstream applications such as real-time PCR and DNA sequencing.
We also describe a primer-probe partial overlap approach which significantly simplified the
melting curve patterns and minimized the influence of viral genome polymorphism on assay
accuracy. Analysis of 62 patient samples showed a complete match of the melting curve
patterns with the sequencing results. More than 97% of HBV BCP sequences in the
GenBank database can be correctly identified by the melting curve analysis. The
combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in
the presence of a 100,000-fold excess of the WT DNA.
http://jcm.asm.org/cgi/content/abstract/49/7/2440
Performance Characteristics and Comparison of Abbott
and artus Real-Time Systems for Hepatitis B Virus DNA
Quantification
Ashrafali M. Ismail1, Jayashree Sivakumar1, Raghavendran Anantharam1, Sujitha
Dayalan1, Prasanna Samuel2, Gnanadurai J. Fletcher1, Manu Gnanamony1 and Priya
Abraham1*
Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of
HBV infection. With several HBV DNA quantification assays available, it is important to
use the most efficient testing system for virological monitoring. In this study, we evaluated
the performance characteristics and comparability of three HBV DNA quantification
systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with
QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the
QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these
systems were established against the WHO international standards for HBV DNA and were
found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients
of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16
to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to
7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were
used for comparison of assays, and paired quantitative results showed strong correlation by
linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artusDSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a
good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10
log10 IU/ml and limits of agreement of –0.91 to 1.11 log10 IU/ml. No genotype-specific bias
was seen in all three systems for HBV genotypes A, C, and D, which are predominant in
this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems
show more comparable performance than the artus-DB system, meeting the current
guidelines for assays to be used in the management of hepatitis B.
http://jcm.asm.org/cgi/content/abstract/49/9/3215
Three Amino Acid Mutations (F51L, T59A, and S390L)
in the Capsid Protein of the Hepatitis E Virus
Collectively Contribute to Virus Attenuation
Laura Córdoba,1 Yao-Wei Huang,1 Tanja Opriessnig,2 Kylie K. Harral,1 Nathan M.
Beach,1 Carla V. Finkielstein,3 Suzanne U. Emerson,4, and Xiang-Jin Meng1*
Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, and
the mechanisms of HEV replication and pathogenesis are largely unknown. We previously
identified an attenuated genotype 3 HEV mutant (pSHEV-1) containing three unique amino
acid mutations (F51L, T59A, and S390L) in the capsid protein. To determine the role of
each of these mutations, we constructed three HEV single mutants (rF51L, rT59A, and
rS390L) which were all found to be replication competent in Huh7 liver cells. To determine
the pathogenicities of the mutants, we utilized the specific-pathogen-free (SPF) pig model
for HEV and a unique inoculation procedure that bypasses the need for propagating
infectious HEV in vitro. A total of 60 pigs were intrahepatically inoculated, via an
ultrasound-guided technique, with in vitro-transcribed full-length capped RNA transcripts
from the infectious clones of each single mutant, the pSHEV-1 triple mutant, wild-type
pSHEV-3, or phosphate-buffered saline (PBS) buffer (n = 10). The results showed that the
F51L mutation partially contributed to virus attenuation, whereas the T59A and S390L
mutations resulted in more drastic attenuation of HEV in pigs, as evidenced by a
significantly lower incidence of viremia, a delayed appearance and shorter duration of fecal
virus shedding and viremia, and lower viral loads in liver, bile, and intestinal content
collected at three different necropsy times. The results indicate that the three mutations in
the capsid protein collectively contribute to HEV attenuation. This study has important
implications for developing a modified live-attenuated vaccine against HEV.
http://jvi.asm.org/cgi/content/abstract/85/11/5338
Standardization of Hepatitis E Virus (HEV) Nucleic Acid
Amplification Technique-Based Assays: an Initial Study
To Evaluate a Panel of HEV Strains and Investigate
Laboratory Performance ,
Sally A. Baylis*, Kay-Martin Hanschmann, Johannes Blümel, C. Micha Nübling on
behalf of the HEV Collaborative Study Group
The performance of hepatitis E virus (HEV) RNA nucleic acid amplification (NAT)-based
assays has been investigated using a panel of HEV-containing plasma samples. The panel
comprised 22 HEV-positive plasma samples representing 10-fold serial dilutions of HEV
genotypes 3a, 3b, 3f, and 4c obtained from blood donors. Two negative-control plasma
samples were included. All samples were blinded. The plasma samples were prepared as
liquid/frozen materials and distributed to participants on dry ice. Laboratories were
requested to test the panel using their routine HEV assays and to score samples as either
positive or negative and could optionally return data in copies/ml for HEV RNA. Twenty
laboratories from 10 different countries participated in the study. Data were returned by all
participating laboratories; 10 laboratories returned quantitative data. All assays except one
were developed in-house using conventional or real-time reverse transcriptase PCR (RTPCR) methodologies. There was a 100- to 1,000-fold difference in sensitivity between the
majority of assays, independent of the virus strain. Although the quantitative data were
limited, for the samples in the range of 6 to 4 log10 copies/ml, the standard deviations of
the geometric means of the samples ranged between 0.38 and 1.09. Except for one
equivocal result, HEV RNA was not detected in the negative samples. The variability of
assay sensitivity highlights the need for the standardization of HEV RNA NAT assays.
http://jcm.asm.org/cgi/content/abstract/49/4/1234
Comparison of Serial Hepatitis C Virus Detection in
Samples Submitted through Serology for Reflex
Confirmation versus Samples Directly Submitted for
Quantitation
Howard B. Gale1*, D. Robert Dufour2, Nazia N. Qazi3,4 and Virginia L. Kan1,4
Using real-time technology, we reliably identified chronic hepatitis C virus (HCV) infection
and quantified virus from reflex samples originally submitted for serologic testing. There
was no need to process specimens obtained directly for quantitation separately. Whether the
initial source is a reflex sample or one obtained directly, a repeat HCV RNA test is needed
before starting treatment.
Hepatitis C Virus Genotypes in Clinical Specimens
Tested at a National Reference Testing Laboratory in the
United States
Jeffrey J. Germer1, Jayawant N. Mandrekar2, Jordan L. Bendel1, P. Shawn Mitchell1
and Joseph D. C. Yao1*
Hepatitis C virus (HCV) genotype (GT) distribution and frequency were studied among
22,407 unique specimens tested at a national reference testing laboratory. Subjects with
HCV GT 3 were younger (P < 0.0001) than those with GT 1, 2, or 4, and the regional
frequencies of HCV GT 2 and 3 ranged from 19.9% to 29.2%.
http://jcm.asm.org/cgi/content/abstract/49/8/3040
Multilaboratory Evaluation of Real-Time PCR Tests for
Hepatitis B Virus DNA Quantification
Angela M. Caliendo1, *, Alexander Valsamakis2, , James W. Bremer3, Andrea
Ferreira-Gonzalez4, Suzanne Granger5, Linda Sabatini6, , Gregory J. Tsongalis7, Yun
F. (Wayne) Wang8, Belinda Yen-Lieberman9, Steve Young10 and Nell S. Lurain3
The performance characteristics of four different assays for hepatitis B virus (HBV)
quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas
AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure
system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range,
reproducibility, and agreement were determined using a serially diluted plasma sample
from a single chronically infected subject. Each assay was tested by at least three
laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0
log10 IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was
approximately 1.5 log10 IU/ml. The linear range spanned 1.0 to at least 7.0 log10 IU/ml for
all assays. Median values were consistently lowest for artus HBV and highest for Cobas
AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction
were the most reproducible; however, the overall variability was minor since the standard
deviations for the means of all tested concentrations were 0.32 log10 IU/ml for all assays.
False-positive results were observed with all assays; the highest rates occurred with tests
using manual nucleic acid extraction. The performance characteristics of these assays
suggest that they are useful for management and therapeutic monitoring of chronic HBV
infection.
http://jcm.asm.org/cgi/content/abstract/49/8/2854
Enhancement of Replication of RNA Viruses by ADAR1
via RNA Editing and Inhibition of RNA-Activated
Protein Kinase
Jean-François Gélinas,1,2, Guerline Clerzius,1,3, Eileen Shaw,1,3, and Anne
Gatignol1,2,3*
Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA binding
protein and RNA-editing enzyme that modifies cellular and viral RNAs, including coding
and noncoding RNAs. This interferon (IFN)-induced protein was expected to have an
antiviral role, but recent studies have demonstrated that it promotes the replication of many
RNA viruses. The data from these experiments show that ADAR1 directly enhances
replication of hepatitis delta virus, human immunodeficiency virus type 1, vesicular
stomatitis virus, and measles virus. The proviral activity of ADAR1 occurs through two
mechanisms: RNA editing and inhibition of RNA-activated protein kinase (PKR). While
these pathways have been found independently, the two mechanisms can act in concert to
increase viral replication and contribute to viral pathogenesis. This novel type of proviral
regulation by an IFN-induced protein, combined with some antiviral effects of hyperediting,
sheds new light on the importance of ADAR1 during viral infection and transforms our
overall understanding of the innate immune response.
http://jvi.asm.org/cgi/content/abstract/85/17/8460
Hepatitis B Virus Genotype C Isolates with Wild-Type
Core Promoter Sequence Replicate Less Efficiently than
Genotype B Isolates but Possess Higher Virion Secretion
Capacity
Yanli Qin,1,2 Xiaoli Tang,1 Tamako Garcia,1 Munira Hussain,3 Jiming Zhang,2* Anna
Lok,3 Jack Wands,1 Jisu Li,1, and Shuping Tong1*
Infection by hepatitis B virus (HBV) genotype C is associated with a prolonged viremic
phase, delayed hepatitis B e antigen (HBeAg) seroconversion, and an increased incidence
of liver cirrhosis and hepatocellular carcinoma compared with genotype B infection.
Genotype C is also associated with the more frequent emergence of core promoter
mutations, which increase genome replication and are independently associated with poor
clinical outcomes. We amplified full-length HBV genomes from serum samples from
Chinese and U. S. patients with chronic HBV infection and transfected circularized genome
pools or dimeric constructs of individual clones into Huh7 cells. The two genotypes could
be differentiated by Western blot analysis due to the reactivities of M and L proteins toward
a monoclonal pre-S2 antibody and slightly different S-protein mobilities. Great variability
in replication capacity was observed for both genotypes. The A1762T/G1764A core
promoter mutations were prevalent in genotype C isolates and correlated with increased
replication capacity, while the A1752G/T mutation frequently found in genotype B isolates
correlated with a low replication capacity. Importantly, most genotype C isolates with wildtype core promoter sequence replicated less efficiently than the corresponding genotype B
isolates due to less efficient transcription of the 3.5-kb RNA. However, genotype C isolates
often displayed more efficient virion secretion. We propose that the low intracellular levels
of viral DNA and core protein of wild-type genotype C delay immune clearance and trigger
the subsequent emergence of A1762T/G1764A core promoter mutations to upregulate
replication; efficient virion secretion compensates for the low replication capacity to ensure
the establishment of persistent infection by genotype C.
http://jvi.asm.org/cgi/content/abstract/85/19/10167
Mutational Analysis of the Hypervariable Region of
Hepatitis E Virus Reveals Its Involvement in the
Efficiency of Viral RNA Replication
R. S. Pudupakam, Scott P. Kenney, Laura Córdoba, Yao-Wei Huang, Barbara A.
Dryman, Tanya LeRoith, F. William Pierson,, and Xiang-Jin Meng*
The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in
ORF1 that tolerates small deletions with respect to infectivity. To further investigate the
role of the HVR in HEV replication, we constructed a panel of mutants with overlapping
deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype
1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA
replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants
were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication
efficiency. To further verify the results, we constructed HVR deletion mutants by using a
genetically divergent, nonmammalian avian HEV, and similar effects on viral replication
efficiency were observed when the avian HEV mutants were tested in LMH cells.
Furthermore, the impact of complete HVR deletion on virus infectivity was tested in
chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion
mutant was still replication competent in LMH cells, the complete HVR deletion resulted in
a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in
sequence and length among different HEV genotypes, we further examined the
interchangeability of HVRs and demonstrated that HVR sequences are functionally
exchangeable between HEV genotypes with regard to viral replication and infectivity in
vitro, although genotype-specific HVR differences in replication efficiency were observed.
The results showed that although the HVR tolerates small deletions with regard to
infectivity, it may interact with viral and host factors to modulate the efficiency of HEV
replication.
http://jvi.asm.org/cgi/content/abstract/85/19/10031
Hepatitis C Virus-Induced Cancer Stem Cell-like
Signatures in Cell Culture and Murine Tumor
Xenografts
Naushad Ali1,*, Heba Allam1, Randal May1, Sripathi M. Sureban1, Michael S. Bronze1,
Ted Bader1, Shahid Umar1, Srikant Anant2, and Courtney W. Houchen1,*
Hepatitis C virus (HCV) infection is a prominent risk factor for the development of
hepatocellular carcinoma (HCC). Similar to most solid tumors, HCCs are believed to
contain poorly differentiated cancer stem-like cells (CSCs) that initiate tumorigenesis and
confer resistance to chemotherapy. In these studies, we demonstrate that expression of HCV
subgenomic replicon in cultured cells results in acquisition of CSC traits. These traits
include enhanced expression of DCAMKL-1, Lgr5, CD133, α-fetoprotein, cytokeratin-19
(CK19), Lin28 and c-Myc. Conversely, curing of the replicon from these cells results in
diminished expression of these factors. The putative stem cell marker, DCAMKL-1, is also
elevated in response to the overexpression of a cassette of pluripotency factors. The
DCAMKL-1-positive cells isolated from hepatoma cell lines by fluorescence activated cellsorting (FACS) form spheroids in matrigel. The HCV RNA abundance and NS5B level is
significantly reduced by the siRNA-led depletion of DCAMKL-1. We further demonstrate
that HCV replicon-expressing cells initiate distinct tumor phenotypes compared to the
tumors initiated by parent cells lacking the replicon. This HCV-induced phenotype is
characterized by high-level expression/co-expression of DCAMKL-1, CK19, α-fetoprotein,
and active c-Src. The results obtained by the analysis of liver tissues from HCV-positive
patients and liver tissue microarray reiterate these observations. In conclusion, chronic HCV
infection appears to predispose cells on the path of acquiring cancer stem cell-like traits by
inducing DCAMKL-1, hepatic progenitor and stem cell-related factors. The DCAMKL-1
also represents a novel cellular target for combating HCV-induced hepatocarcinogenesis.
http://jvi.asm.org/cgi/content/abstract/JVI.05920-11v1
Hepatocytes traffic and export hepatitis B virus
basolaterally by polarity-dependent mechanisms
Purnima Bhat1,2,3,*, Michelle J. Snooks1, and David A. Anderson1,3,4
Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral
export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either
non-polarized, or have morphology unsuitable for the study of viral export. Here, we
investigate the mechanisms of trafficking and export for the hepadnaviruses, hepatitis B
virus (HBV) and duck hepatitis B virus (DHBV), in polarized hepatocyte-derived cell lines
and primary duck hepatocytes. DHBV export, but not replication, was dependent on the
development of hepatocyte polarity, with export significantly abrogated over time as
primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and
Adenovirus-based transduction, we observed that export of both HBV and DHBV was
vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and
non-polarized C11 cells during persistent, long-term DHBV infection demonstrated that
newly synthesized sphingolipid and virus displayed significant co-localization and FRET,
implying co-transportation from the Golgi to the plasma membrane. Notably, 15% of virus
was released apically from polarized cells, corresponding to secretion into the bile duct in
vivo, also in association with sphingolipids. We conclude that DHBV, and probably HBV,
is reliant upon hepatocyte polarity to be efficiently exported, and this export is in
association with sphingolipid structures, possibly lipid rafts. This study provides novel
insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with
potential relevance to pathogenesis and immune tolerance.
http://jvi.asm.org/cgi/content/abstract/JVI.05344-11v1
Cell-to-Cell Contact with Hepatitis C Virus-Infected
Cells Reduces Functional Capacity of Natural Killer
Cells
Joo Chun Yoon1,2, Jong-Baeck Lim3, Jeon Han Park1,2, and Jae Myun Lee1,2,*
The distinct feature of hepatitis C virus (HCV) infection is high incidence of chronicity.
The reason for chronic HCV infection has been actively investigated, and impairment of
innate and adaptive immune responses against HCV is proposed as a plausible cause.
Whereas functional impairment of HCV-specific T cells is well characterized, the role and
functional status of natural killer (NK) cells in each phase of HCV infection are still elusive.
We therefore investigated whether direct interaction between NK cells and HCV-infected
cells modulates NK cell function. HCV-permissive human hepatoma cell lines were
infected with cell-culture-generated HCV virions and cocultured with primary human NK
cells. Cell-to-cell contact between NK cells and HCV-infected cells reduced NK cells'
capacity to degranulate and lyse target cells, especially in the CD56dim NK cell subset. The
decrease in degranulation capacity was correlated with downregulated expression of NK
cell activating receptors such as NKG2D and NKp30 on NK cells. The ability of NK cells
to produce and secrete interferon (IFN)- also diminished after exposure to HCV-infected
cells. The decline of IFN- production was consistent with the reduction of NK cell
degranulation. In conclusion, cell-to-cell contact with HCV-infected cells negatively
modulated functional capacity of NK cells, and the inhibition of NK cell function was
associated with downregulation of NK activating receptors on NK cell surfaces. These
observations suggest that direct cell-to-cell interaction between NK cells and HCV-infected
hepatocytes may impair NK cell function in vivo and thereby contribute to the
establishment of chronic infection.
http://jvi.asm.org/cgi/content/abstract/JVI.00838-11v1
Comparison of a newly developed automated and
quantitative hepatitis C virus (HCV) core antigen test
with the HCV RNA assay for the clinical usefulness of
confirming anti-HCV results.
Recep Kesli1, Hakk Polat2, Yuksel Terzi3, Muhammet Guzel Kurtoglu1 and Yavuz
Uyar4
HCV is a global healthcare problem. Diagnosis of HCV infection is mainly based on the
detection of anti-HCV antibodies as a screening test on sera samples. Recombinant
immunoblot assays are used as supplemental tests and in the final detection and
quantification of HCV RNA in confirmatory tests.
In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for
confirming anti-HCV results to determine whether the HCV core antigen test may be used
as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values
of the total HCV core antigen test by determining the diagnostic specificity and sensitivity
rates compared with the HCV RNA test.
A total of 212 treatment-naive patients provided serum were analysed for anti-HCV and
HCV core antigen assay, both with Abbott Architect, and the molecular HCV RNA assay is
a confirmatory test by using a reverse transcription-polymerase chain reaction method. The
diagnostic sensitivity, specificity and the positive and negative predictive values of the
HCV core antigen assay compared to the HCV RNA test were 96.3 %, 100 %, 100 %, and
89.7 %, respectively. The levels of HCV core antigen showed a good correlation with those
from the HCV RNA quantification (r=0.907).
In conclusion, the Architect HCV Ag assay is highly specific, sensitive, reliable, easy to
perform, reproducible, cost-effective and applicable as a screening, supplemental and preconfirming test for anti-HCV assays in the laboratory procedures used for the diagnosis of
hepatitis C virus infection.
http://jcm.asm.org/cgi/content/abstract/JCM.05292-11v1
Hepatitis C Virus Infection Is Blocked by HMGB1
Released from Virus-Infected Cells
Jong Ha Jung,1 Ji Hoon Park,1 Min Hyeok Jee,1 Sun Ju Keum,1 Min-Sun Cho,2 Seung
Kew Yoon,3, and Sung Key Jang1,4,5*
High-mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host
immune responses, is an endogenous danger signal involved in the pathogenesis of various
infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here,
we show that HMGB1 protein is translocated from the nucleus to cytoplasm and
subsequently is released into the extracellular milieu by HCV infection. Secreted HMGB1
triggers antiviral responses and blocks HCV infection, a mechanism that may limit HCV
propagation in HCV patients. Secreted HMGB1 also may have a role in liver cirrhosis,
which is a common comorbidity in HCV patients. Further investigations into the roles of
HMGB1 in the diseases caused by HCV infection will shed light on and potentially help
prevent these serious and prevalent HCV-related diseases.
HepG2 cells expressing miR-122 support the entire
hepatitis C virus life cycle
Christopher M. Narbus1, Benjamin Israelow1, Marion Sourisseau1, Maria L. Michta1,
Sharon E. Hopcraft1, Gusti M. Zeiner2, and Matthew J. Evans1,**
The liver specific microRNA, miR-122, is required for efficient hepatitis C virus (HCV)
RNA replication in both cell culture and in vivo. In addition, nonhepatic cells have been
rendered more efficient at supporting this stage of the HCV life cycle by miR-122
expression. This study investigated how miR-122 influences HCV replication in the miR122 deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted
efficient HCV RNA replication and infectious virion production. When a missing HCV
receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV
life cycle.
http://jvi.asm.org/cgi/content/abstract/JVI.05843-11v1
Roles of the Envelope Proteins in the Amplification of
cccDNA and Completion of Synthesis of the Plus-Strand
DNA in Hepatitis B Virus
Thomas B. Lentza,b, and Daniel D. Loeba,*
Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is
synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is
derived from RC DNA from the infecting virion, but additional copies of cccDNA are
derived from newly synthesized RC DNA molecules in a process termed intracellular
amplification. It has been shown that the large viral envelope protein limits intracellular
amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in
regulating amplification of cccDNA in HBV is not well characterized. The present report
demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation
of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA.
Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level
of cccDNA, which inversely correlated with the level of the envelope proteins. We found
that expression of L protein alone or in combination with M or S proteins led to a decrease
in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Co-expression
of L and M led to greater regulation than either L alone or L and S. Co-expression of all
three envelope proteins was also found to limit completion of plus-strand DNA synthesis
and the degree of this effect correlated with the level of the proteins and virion secretion.
Hepatitis C Virus Stimulates the Phosphatidylinositol 4Kinase III Alpha-Dependent Phosphatidylinositol 4Phosphate Production That Is Essential for Its
Replication
Kristi L. Berger, Sean M. Kelly, Tristan X. Jordan, Michael A. Tartell,, and Glenn
Randall*
Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus
(HCV) replication. We initiated this study to determine whether HCV directly engages
PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely
required for HCV replication using a small interfering RNA transcomplementation assay.
Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in
phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which
partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P
accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed
after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or
RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV
polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced
cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein
expression was precluded by silencing the expression of PI4KA, but not the related PI4KB.
Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins,
including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P
production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA
coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected
293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent
PI4P production influences replication complex formation.
http://jvi.asm.org/cgi/content/abstract/85/17/8870
Peptidyl-Prolyl Isomerase Pin1 Is a Cellular Factor
Required for Hepatitis C Virus Propagation
Yun-Sook Lim, Huong T. L. Tran, Soo-Je Park, Seung-Ae Yim,, and Soon B.
Hwang*
The life cycle of hepatitis C virus (HCV) is highly dependent on cellular factors. Using
small interfering RNA (siRNA) library screening, we identified peptidyl-prolyl cis-trans
isomerase NIMA-interacting 1 (Pin1) as a host factor involved in HCV propagation. Here
we demonstrated that silencing of Pin1 expression resulted in decreases in HCV replication
in both HCV replicon cells and cell culture-grown HCV (HCVcc)-infected cells, whereas
overexpression of Pin1 increased HCV replication. Pin1 interacted with both the NS5A and
NS5B proteins. However, Pin1 expression was increased only by the NS5B protein. Both
the protein binding and isomerase activities of Pin1 were required for HCV replication.
Juglone, a natural inhibitor of Pin1, inhibited HCV propagation by inhibiting the interplay
between the Pin1 and HCV NS5A/NS5B proteins. These data indicate that Pin1 modulates
HCV propagation and may contribute to HCV-induced liver pathogenesis.
http://jvi.asm.org/cgi/content/abstract/85/17/8777
Development of a Second Version of the Cobas
AmpliPrep/Cobas TaqMan Hepatitis C Virus
Quantitative Test with Improved Genotype Inclusivity
Johannes Vermehren1, Giuseppe Colucci2, Peter Gohl3, Nabila Hamdi4, Ahmed Ihab
Abdelaziz4, Ursula Karey1, Diana Thamke2, Heike Zitzer2, Stefan Zeuzem1 and
Christoph Sarrazin1*
Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of realtime PCR-based assays with low limits of detection and broad dynamic ranges for
quantification. In the present study, the performance of two second-version prototypes of
the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with
decreased sample input volume and improved genotype inclusivity was investigated. A total
of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype
1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were
processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM),
Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype
formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4
transcripts containing rare variant sequences were tested. The mean log10 HCV RNA
differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to
CAM were –0.05, 0.05, –0.12, –0.10, –0.44, and –0.29 for patients with GT1, GT2, GT3,
GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including
isolates with known variants within the 5' untranslated region (G145A, A165T) that were
underquantified with CAP/CTM were correctly quantified with the second-version
prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts
with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA
across all HCV genotypes, including specimens with rare polymorphisms previously
associated with underquantification.
http://jcm.asm.org/cgi/content/abstract/49/9/3309
Allele-Specific Real-Time PCR System for Detection of
Subpopulations of Genotype 1a and 1b Hepatitis C NS5B
Y448H Mutant Viruses in Clinical Samples
Andrew S. Bae, Karin S. Ku, Michael D. Miller, Hongmei Mo and Evguenia S.
Svarovskaia*
The Y448H mutation in NS5B has been selected by GS-9190 as well as several
benzothiadiazine hepatitis C virus (HCV) polymerase inhibitors in vitro and in vivo.
However, the level and the evolution kinetics of this resistance mutation prior to and during
treatment are poorly understood. In this study, we developed an allele-specific real-time
PCR (AS-PCR) assay capable of detecting Y448H when it was present at a level down to
0.5% within an HCV population of genotype 1a or 1b. No Y448H mutation was detected
above the assay cutoff of 0.5% in genotype 1b-infected Con-1 replicons prior to in vitro
treatment. However, the proportion of replicons with the Y448H mutation rapidly increased
in a dose-dependent manner upon treatment with GS-9190. After 3 days of treatment, 1.2%,
6.8%, and >50% of the replicon population expressed Y448H with the use of GS-9190 at 1,
10, and 20 times its 50% effective concentration, respectively. In addition, plasma from 65
treatment-naïve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively)
was tested for the presence of Y448H by AS-PCR and population sequencing. As expected,
all patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR
results were obtained for 62/65 samples tested, with low levels of Y448H ranging from
0.5% to 3.0% detected in 5/62 (8%) treatment-naïve patient samples. These findings suggest
the need for combination therapy with HCV-specific inhibitors to avoid viral rebound of
preexisting mutant HCV.
http://jcm.asm.org/cgi/content/abstract/49/9/3168
Comparison of a Novel Real-Time PCR Assay with
Sequence Analysis, Reverse Hybridization, and Multiplex
PCR for Hepatitis B Virus Type B and C Genotyping
Yao Zhao1,2, , Xiu-Yu Zhang2,5, , Yuan Hu2, Wen-Lu Zhang2, Jie-Li Hu2, Ai-Zhong
Zeng3, Jin-Jun Guo4, Wen-Xiang Huang3, Wei-Xian Chen5, You-Lan Shan4 and AiLong Huang2*
We compared a novel real-time genotyping and quantitative PCR (GQ-PCR) assay, direct
sequence analysis, reverse hybridization, and multiplex PCR for genotyping hepatitis B
virus (HBV) in 127 HBV-infected patients. We found that GQ-PCR had the highest
concordance with sequence analysis and the highest detection rate for mixed genotype
detecting.
http://jcm.asm.org/cgi/content/abstract/49/9/3392
Discovery of Potent Hepatitis C Virus NS5A Inhibitors
with Dimeric Structures
Julie A. Lemm1, John E. Leet2, Donald R. O'Boyle, II1, Jeffrey L. Romine3, Xiaohua
Stella Huang4, Daniel R. Schroeder4, Jeffrey Alberts5, , Joseph L. Cantone4, Jin-Hua
Sun1, Peter T. Nower1, Scott W. Martin3, Michael H. Serrano-Wu3, , Nicholas A.
Meanwell3, Lawrence B. Snyder3, and Min Gao1,*
The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the
50% effective concentration (EC50) of the initial lead, the thiazolidinone BMS-824, was 10
nM in the replicon assay, it underwent transformation to other inhibitory species after
incubation in cell culture medium. The biological profile of BMS-824, including the EC50,
the drug concentration required to reduce cell growth by 50% (CC50), and the resistance
profile, however, remained unchanged, triggering an investigation to identify the
biologically active species. High-performance liquid chromatography (HPLC) biogram
fractionation of a sample of BMS-824 incubated in medium revealed that the most active
fractions could readily be separated from the parental compound and retained the biological
profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active
species was determined to be a dimer of BMS-824 derived from an intermolecular radicalmediated reaction of the parent compound. Based upon an analysis of the structural
elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative
BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and
showed a resistance profile similar to that of BMS-824, with changes in compound
sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been
crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential
mode of inhibition of NS5A to be discussed. Identification of the stable, active
pharmacophore associated with these NS5A inhibitors provided the foundation for the
design of more potent inhibitors with broad genotype inhibition. This culminated in the
identification of BMS-790052, a compound that preserves the symmetry discovered with
BMS-346.
http://aac.asm.org/cgi/content/abstract/55/8/3795
Multicentric Evaluation of New Commercial Enzyme
Immunoassays for the Detection of Immunoglobulin M
and Total Antibodies against Hepatitis A Virus
M. C. Arcangeletti1,*, E. Dussaix2, F. Ferraglia1, A. M. Roque-Afonso2, A. Graube2
and C. Chezzi1
A multicentric clinical study was conducted on representative sera from 1,738 European
and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays
from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the
good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting
total antibodies and IgM, respectively).
http://cvi.asm.org/cgi/content/abstract/18/8/1391
Temporal Variations in the Hepatitis C Virus Intrahost
Population during Chronic Infection
Sumathi Ramachandran, David S. Campo, Zoya E. Dimitrova, Guo-liang Xia,
Michael A. Purdy,, and Yury E. Khudyakov*
The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms
responsible for the establishment of chronic infections and to development of a vaccine and
therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1
and NS5A, were sequenced from four treatment-naïve chronically infected patients who
were followed up from the acute stage of infection for 9 to 18 years. Median-joining
network analysis indicated that the majority of the HCV intrahost variants were observed
only at certain time points, but some variants were detectable at more than one time point.
In all patients, these variants were found organized into communities or subpopulations. We
hypothesize that HCV intrahost evolution is defined by two processes: incremental changes
within communities through random mutation and alternations between coexisting
communities. The HCV population was observed to incrementally evolve within a single
community during approximately the first 3 years of infection, followed by dispersion into
several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of
the observation period, while HCV variants in the other two patients converged into another
single subpopulation after 9 to 12 years of dispersion. The final subpopulation in these two
patients was under purifying selection. Intrahost HCV evolution in all four patients was
characterized by a consistent increase in negative selection over time, suggesting the
increasing HCV adaptation to the host late in infection. The data suggest specific staging of
HCV intrahost evolution.
http://jvi.asm.org/cgi/content/abstract/85/13/6369