text:
RNA interference for viral infections.
ABSTRACT The treatment of viral infections has relied on pre-emptive vaccination or use of a limited range of anti-viral drugs. However, the majority of viruses have no available drugs and treatment is merely supportive. RNA interference (RNAi) offers the ability to directly and rapidly treat virus infections via the targeting of viral genes. Indeed, clinical trials have already been undertaken with promising results. Here we review the current state of the RNAi field for the treatment of viral infections such as HIV, human papillomavirus and HCV. We also review novel strategies including the concept of targeting self-genes to limit viral infection and activating the immune system for improved outcomes. Finally we examine innovative approaches being pursued at the Australian Infectious Diseases Research Centre including the use of highthroughput siRNA screens to identify new antiviral targets.
Article: In vivo comparison of local versus systemic delivery of immunostimulating siRNA in HPV-driven tumours.[Show abstract] [Hide abstract]
ABSTRACT: Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies.Immunology and Cell Biology advance online publication, 12 November 2013; doi:10.1038/icb.2013.75.Immunology and Cell Biology 11/2013; · 3.93 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic.The Open Virology Journal 01/2012; 6:204-15.
Article: Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives.[Show abstract] [Hide abstract]
ABSTRACT: The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.BioMed Research International 01/2014; 2014:161794. · 2.71 Impact Factor
Page 1
Current Drug Targets, 2012, 13, 1411-1420 1411
RNA Interference for Viral Infections
Stephen J. Blake, Fawzi F. Bokhari and Nigel A.J. McMillan*
Australian Infectious Diseases Research Centre and Diamantina Institute, University of Queensland, Brisbane, QLD
4072, Australia
Abstract: The treatment of viral infections has relied on pre-emptive vaccination or use of a limited range of anti-viral
drugs. However, the majority of viruses have no available drugs and treatment is merely supportive. RNA interference
(RNAi) offers the ability to directly and rapidly treat virus infections via the targeting of viral genes. Indeed, clinical trials
have already been undertaken with promising results. Here we review the current state of the RNAi field for the treatment
of viral infections such as HIV, human papillomavirus and HCV. We also review novel strategies including the concept of
targeting self-genes to limit viral infection and activating the immune system for improved outcomes. Finally we examine
innovative approaches being pursued at the Australian Infectious Diseases Research Centre including the use of high-
throughput siRNA screens to identify new antiviral targets.
Keywords: HCV, HIV, HPV, RNA interference, screening.
over the microbial world, at least over bacteria, and has
saved countless millions of lives, we have not had the same
success when it comes to viruses. The nature of viruses as
intracellular parasites which have evolved to be completely
reliant on host cells for their replication has meant that de-
veloping virus-specific drugs represents a unique challenge.
Indeed, one only has to look at the short list of currently
available antivirals to realise that this is an area of need and
one in which we have had only modest success. Even with
the resources and effort that went into developing anti-HIV
therapies we have only managed to turn this previously un-
treatable infection into one that is chronic and clinically
manageable. It is sobering to realise that no cure for HIV
appears likely in the near future.
Viruses have not had it all their own way of course. Im-
munisation is clearly the most successful means developed to
date to combat viral infections and this has led to the com-
plete eradication of smallpox [2], hopefully soon to be fol-
lowed by polio, although the latter is proving more difficult
[3]. The recent introduction of the vaccine against cervical
cancer, a disease caused by human papillomavirus, is the
latest example of viral vaccine success with the most recent
data suggesting that the rates of pre-cancerous lesions are
falling in target populations since its introduction [4, 5].
However, vaccines do not exist for most viral infections.
Moreover, there are situations where immunisation is either
inadequate or has proven to be ineffective, or in some cases
dangerous. For example, vaccines against HIV have not been
successful despite enormous efforts and resources [6]. Early
RSV vaccines generated poor immune responses that not
only did not prevent infection, but made subsequent infec-
tions more severe [7]. Furthermore, vaccination is ineffective
*Address correspondence to this author at the School of Medical Science,
Griffith University, Gold Coast Campus, QLD 4222, Qld, Australia;
Tel: 07 5552 7135; E-mail: email@example.com
While the advent of antibiotics heralded a major victory
for viruses that replicate actively in sites of immune privi-
lege, such as the central nervous system (e.g. Herpes virus
family), or attack the immune system itself (Human Immu-
nodeficiency Virus {HIV}).
Anti-viral drugs provide an option against viruses with-
out effective vaccines and effective anti-viral drugs are
available for a very limited number of viruses. Effective anti-
viral therapies against HIV have vastly improved treatment
options as while not curing the virus, allow it to become a
clinically manageable chronic infection and increasing pa-
tient survival drastically. Anti-influenza medications such as
Tamiflu® (Oseltamivir) and Relenza® (Zanamivir) have re-
cently been developed [8] and work by blocking the viral
gene neuraminidase, effectively blocking viral spread by
preventing viral particles budding from infected cells. These
drugs are useful in the acute hospital setting but are limited
in general use due to the inability to rapidly identify patients
infected with Influenza rather than a range of other respira-
tory viruses and the fact that patients often present to physi-
cians at a late stage of infection where viral replication is
already being controlled by an immune response.
While much effort has been made to develop new antivi-
rals via natural product or small molecule screens, success
has been time-consuming, fortuitous and difficult to obtain.
With many viruses hijacking host genes for replication de-
signing specific anti-viral drugs has proven to be difficult.
Clearly new approaches are required to allow us to rapidly
identify novel antiviral drug targets not only within the virus
itself but also within the host cell and it is here that RNA
interference offers exciting new possibilities. RNA interfer-
ence, or RNAi, is a means by which one can achieve the si-
lencing of single genes within a cell via the introduction of
short-interfering RNA (siRNA) or short-hairpin RNA
(shRNA) [9, 10]. RNAi was discovered in the mid-1990s
firstly in the plant field where Jorgensen described co-
suppression or post-translational gene silencing [11]. The
mechanisms were later expanded upon and explained inde-
1???-????/12 $58.00+.00 © 2012 Bentham Science Publishers
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1412 Current Drug Targets, 2012, Vol. 13, No. 11
Blake et al.
pendently by the laboratories of Baulcombe and Waterhouse
[12, 13]. While initially thought to be limited to plants, it
was soon discovered by Fire and Mello that RNAi would
work in nematodes [10], a discovery for which they were
awarded the Nobel Prize for medicine in 2006. The impor-
tance of RNA interference in the viral setting was quickly
realised with the discovery that exogenously delivered syn-
thetic siRNA was a powerful means to reduce viral gene
expression, with HIV one of the first examples of how RNAi
can work to treat human disease [14]. It offered a viral-
specific therapy to limit or prevent infection. Indeed it has
been speculated that the evolutionary role of RNAi was in
the cellular defence against viruses and the silencing of
transposable elements by the generation of virus-specific
siRNAs [15].
velop RNAi as a direct therapy against viral infections as
well as cancer caused by viral infections. We will also re-
view efforts using RNAi in genome-wide gene expression
studies to identify previously unthought-of and novel anti-
viral drug targets. These screens may allow the development
of new classes of anti-viral drugs as well as to re-purpose
some drugs currently in the clinic but for which an anti-viral
use has not previously been realised.
In this review we will outline the current efforts to de-
2. RNAi AS A THERAPY FOR VIRUSES
pendence on a limited set of viral genes, high levels of repli-
cation and targets that often are very different to human
genes. Additionally, the development time for new siRNA is
short compared to other specific drugs, such as small mole-
cule inhibitors, meaning siRNA against newly isolated vi-
ruses or to combat siRNA resistant mutations can be rapidly
developed. An alternative strategy to targeting viral genes is
to target host genes that are critical for viral lifestyle, such as
surface receptors required for cell entry (e.g. CCR5 in HIV)
or host genes involved in intracellular viral replication.
RNAi is ideal for use against viruses due to viruses' de-
of many viruses in vitro, the efforts to design in vivo and
clinical therapies have been less successful. The main issues
preventing effective in vivo therapies revolve around effec-
tive delivery to target cells, an issue that is seen as the major
obstacle to developing clinical siRNA. The liver is an excel-
lent target for siRNA therapy with many different groups
showing siRNA-loaded nanoparticles distribute in high con-
centrations to the liver following intravenous administration.
This has encouraged development of siRNA therapies for
HCV, HBV as well as various liver cancers and cholesterol
metabolism. Respiratory viruses have been shown to be well
controlled by intranasal delivery of siRNA in mice, with
delivery here beneficial in that it avoids potential complica-
tions following systemic drug delivery. SiRNA has been
shown to be effective at treating a diverse range of viruses
(Table 1). There are two possible strategies for using siRNA
as a therapy for viruses - direct targeting of viral genes or
downregulation of host genes required for virus replication.
While RNAi has been potent at blocking the replication
2.1. Direct Inhibition of Viral Genes by siRNA
ful, with many viruses including the Respiratory Syncytial
RNAi inhibition of viral genes has been highly success-
Virus (RSV), Hepatitis B Virus (HBV), HIV, and influenza
virus able to be strongly inhibited in cell culture and small
animal models. The targets generally included genes critical
for viral survival, replication, or cell infection. Unlike con-
ventional drugs, RNAi can target any genes, including previ-
ously undruggable proteins such as transcription factors.
Translation of these successes to clinical trials has been
slow, with two trials recently completed and numerous more
underway as discussed below.
Table 1. Examples of siRNAs used to Treat Virus Infections.
Virus Genes targeted References
Influenza A
Acidic polymerase
SARS coronavirus
Spike protein
M protein
Human Papilloma
Epstein Barr Virus
[26, 27]
Ebola Virus
Polymerase (L) gene
[28, 29]
Respiratory Syncytial Virus
siRNA were targeting RSV [30-32]. RSV infection is a ma-
jor cause of seasonal colds, which contribute a large cost to
the public health system and can be deadly to patients with
compromised immune systems, such as the elderly or infants
[33, 34]. With no vaccine, it is also ideal to test novel thera-
pies such as siRNA. RSV represents the most developed
study of RNAi against viruses, with the completion of a
phase 2 clinical trial. The results of two animal studies were
published in 2005 using different approaches to treat RSV
[30, 31]. The first by Zhang et al. [30] used siRNA to target
the viral gene NS1 which is essential for robust viral replica-
tion [35], and has been implicated in dampening host im-
mune responses to virus [36]. In this study, RNAi was in the
form of shRNAs encoded on plasmids and delivered by
lipofectamine, and was shown to inhibit viral replication in
cultures. When delivered intranasally in mice by nanoparti-
cles (TransGenex Nanobiotech Inc), viral replication and
disease was limited in both prophylactic and therapeutic
manners, with increased anti-viral immune responses ob-
served. The second mouse study used siRNAs against RSV-
P protein that were designed in an earlier in vitro study [32].
This study delivered siRNA intranasally before challenge
with RSV and was able to show a significant reduction in
viral load and clinical symptoms in the mice [31]. There was
also a therapeutic effect, as delivering siRNA up to 3 days
post-infection still caused a decrease in symptoms and viral
load. Most siRNA were delivered using TransIT-TKO vec-
tor, however they were surprisingly able to show naked
siRNA to have comparable efficacy. Additionally the results
were seen without the induction of IFN-? and were not ob-
served to inhibit a related Pneumovirus, leading to the con-
Some of the earliest and most promising studies using
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RNA Interference for Viral Infections Current Drug Targets, 2012, Vol. 13, No. 11 1413
clusion that direct inhibition of viral genes and not immune
activation was the cause for virus inhibition.
Anylam of AALN-RSV01, a 19 b.p. siRNA which targets
the RSV nucleocapsid-protein gene and potently blocks RSV
replication in vitro. After passing phase 1 clinical trials [37]
the group extended into a phase 2 clinical trial to study the
drug's efficacy [38]. The siRNA was delivered via saline
inhalation with most study participants receiving multiple
doses from day -1 through to +3 relative to challenge with
RSV culture. While the siRNA treatment had a modest effect
on reducing the total number of patients infected, it had no
significant impact on viral load or any of the measured pa-
tient symptoms. The delivery of this siRNA may be key as
naked siRNA was used, possibly being trapped in the upper
airways, unable to reach the viral infected cells. While naked
siRNA was shown effective in mouse models [31], the respi-
ratory tract of humans is significantly different than rodents,
who are obligate nasal breathers. Despite the very modest
therapeutic effect, this study has demonstrated that nasally
administered siRNA are well tolerated in humans and can
have an impact on respiratory viral growth and Anylam has
recently initiated a multi-centre phase 2b study in RSV-
infected lung transplant patients to further study the drug.
The successes of these studies led to the development by
Hepatitis B Virus
HBV is still a very common chronic infection, with 350 mil-
lion people infected worldwide, and is a leading cause of the
liver cancer hepatocellular carcinoma [40]. HBV replicates
almost exclusively within hepatocytes, an ideal target for
siRNA therapy as liver uptake of siRNA encapsulated in
liposomes or other nanoparticles is extremely high [41, 42].
Early results demonstrated that HBV replication in culture
[43] or in a mouse model [44] was strongly inhibited by
siRNA directed against HBV core and surface antigen genes,
respectively. Some of the most promising anti-HBV siRNA
delivery has been seen with a modified lipid know as a stable
nucleic acid lipid particle (SNALP), where the addition of
modifications including PEG to the lipid formulation re-
sulted in a longer half-life in serum than normal lipids and is
specifically designed to deliver siRNA to the liver [45].
Combined with the SNALP technology, the same group de-
signed siRNA that were chemically modified to improve in
vivo stability [46]. By replacing all the 2'–OH groups of the
siRNA with 2'F, 2'O-Me or 2'H groups the researchers
found a drastic improvement in siRNA serum stability. The
addition of ribonucleotides to 5'-antisense strand of the
siRNA also improved the siRNA potency.
Testing both modified and unmodified siRNA encapsu-
lated in SNALPs, the group administered therapy i.v. in a
mouse model of HBV [46]. The results were highly promis-
ing, with multiple doses able to induce a >1 log reduction in
circulating HBV DNA. Long term reduction in viral titres
out to 7 days was observed following a single treatment, and
the group was also able to maintain viral suppression when
dosing mice weekly with siRNA. The modified siRNA were
also shown to have an improved toxicity profile and did not
induce innate immune responses when tested against un-
modified siRNA of the same sequence. Stable delivery of
Despite the availability of a preventative vaccine [39],
shRNA by adeno-associated viruses has been proposed as an
alternative delivery and has been shown to be safe and effec-
tive at reducing HBV viral load in vivo [47, 48]. While these
vectors may achieve more stable gene knockdown, safety
concerns compared to non-viral vectors may limit their clini-
cal use. All the aforementioned studies used HBVsAg as a
target. Overall, siRNA against HBV seems an attractive ap-
proach, however clinical development has been slow, with
only a phase I trial conducted to date [49]. In this trail plas-
mid encoded shRNA against 4 different HBV subtypes was
delivered to patients safely however showed only a modest
effect against HBV. There was also evidence of an anti
siRNA immune response in the patients receiving siRNA
and issues with the company may limit further development
of this siRNA.
2.2. SiRNA Against Human Targets
ruses have high rates of mutations so can quickly develop
resistance against siRNA. While care is required when tar-
geting host genes as to not disrupt any critical normal func-
tions, this field is blossoming due to recent successes of
siRNA inhibition of HIV and Hepatitis C virus (HCV), two
of the most serious chronic viral infections.
Targeting human genes is an attractive option when vi-
Hepatitis C Virus
novel in the antiviral field. Like HBV, HCV is a chronic
viral infection of the liver, with an estimated 200 million
people infected worldwide and an increasing cause of hepa-
tocellular carcinoma [50]. HCV has a very complicated life-
cycle and the discovery of microRNA uncovered a novel
host target to inhibit viral replication. Early gene expression
studies showed a high level of expression of the microRNA,
mir-122, in the liver [51], and this was implicated in lipid
metabolism and cholesterol production [52, 53]. Interest-
ingly, a later study identified an interaction between mir-122
and the 5' non-coding region of HCV with inhibition of mir-
122 drastically reduced HCV replication [54]. Following on
from early results, a subsequent group demonstrated that
inhibition of mir-122 was effective in reducing cholesterol
levels in non-human primates [55]. This study used a 15 b.p.
locked-nucleic-acid-modified oligonucleotide (LNA-antimir)
with the drug name Miravirsen, (Santanis). Using this tech-
nology they demonstrated better liver uptake and activity
compared to a conventional siRNA. The LNA compound
was delivered naked, simplifying siRNA administration as
no delivery vector was required. In studies in chronically
infected chimpanzees, dosing of 1 mg/kg or 5 mg/kg showed
a large reduction in circulating HCV viral titres that re-
mained low following drug withdrawal [56]. Cholesterol
levels were also reduced in the animals, suggesting specific
mir-122 inhibition was being observed, and miravisen was
well tolerated in all groups. The binding site of mir-122 is
conserved across all HCV strains [56], suggesting mir-122
therapy should work in most HCV infections. Miravisen is
currently being tested for safety and efficacy in a double
blind placebo controlled phase 2a clinical trial. The results of
this study are highly anticipated and may demonstrate the
first major clinical success of the RNAi field.
The development of siRNA for HCV is one of the most
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1414 Current Drug Targets, 2012, Vol. 13, No. 11
Blake et al.
Human Immunodeficiency Virus
replication in cultures [57, 58], it was also shown that the
virus could rapidly escape siRNA inhibition through muta-
tions altering the siRNA binding site [59]. Like combina-
tional retroviral therapy, successful inhibition of HIV by
siRNA is likely to be only achievable by the simultaneous
administration of multiple siRNAs [60]. Due to this escape
phenomena an attractive alternative approach to inhibit of
HIV is to target cell surface receptors which mediate viral
entry, such as CD4 and CCR5 [61, 62]. Indeed, homozygotes
for a mutation of CCR5 known as the CCR5?32 allele have
been shown to have drastically reduced viral susceptibility
within an at risk population [63]. Recently the bone marrow
transplantation of a HIV-positive patient using marrow from
a CCR5?32 donor reduced viral load to a level considered
curative [64], indicating inhibition of CCR5 is a very attrac-
tive strategy. Using either siRNA or lentiviral encoded
shRNA a number of groups were able to show HIV infection
to be inhibited in macrophages and T-cells via CCR5 or CD4
downregulation. While promising results have been observed
using aptamers or an anti-CD7 antibody-peptide siRNA con-
jugate to target and inhibit HIV and CCR5 in T-cells in in
vitro studies and humanized mouse models [65, 66], the most
well progressed therapy has used a lentivirus vector to de-
liver shRNA to bone marrow cells before transplantation.
Lentiviral vectors are an attractive therapy for HIV patients
as they can enter haematopoietic stem cells and can express
shRNA permanently. The first clinical trial of such a strategy
has come from an American group who developed a lentivi-
ral construct that used shRNA to inhibit CCR5 expression
and additionally the tat/rev genes of the virus and a decoy
TAR sequence to inhibit HIV polymerase [67]. To deliver
this lentiviral construct, the group took advantage of a hae-
matopeitic stem cell transplantation that was being under-
taken to treat patients for AIDS related lymphoma [68]. In
this setting patients receive bone marrow transplantations,
often of their own cryopreseved CD34+ stem cells, after
whole body irradiation to eradicate the lymphoma. A tiny
proportion (0.2%) of cells were transduced with the lentiviral
construct due to safety concerns about the impact of the len-
tivirus on normal cell function, meaning the impact of the
therapy on viral replication was unable to be assessed. How-
ever, the group was able to demonstrate long term engraft-
ment and expression of the lentivirus in both bone marrow
progenitors and blood T-cells, B-cells, and macrophages up
to 2 years after engraftment, demonstrating the feasibility of
the procedure to safely block viral replication. While a bone
marrow transplant is a dangerous and costly procedure, the
ability to perform the therapy with a patient's own cells and
potential for a cure to HIV means lentiviral delivered shRNA
therapy is one of the most promising for AIDS treatment.
While silencing of HIV genes was able to reduce viral
2.3. Immune Activation by siRNA
established that siRNA can activate innate immune responses
through detection via pattern recognition receptors and lead
to production of type 1 interferons IFN-? and IFN-? [69,
70]. Given IFNs' natural antiviral activity, the concept of
using this feature of siRNAs to bolster these effects has been
explored by several groups including our own. While it has
While overlooked in some early studies, it is now well
been shown that siRNAs can be modified to activate recep-
tors such as Rig-1 [71] or toll like receptor TLR-9 [72],
siRNA is normally recognised by TLR-7 in mice and TLR-7
and 8 in humans [70]. These pattern recognition receptors
are normally involved in the detection of foreign viral and
bacterial nucleic acids. The recognition of TLRs by siRNA is
sequence specific, with certain sequences such as those
which are uridine-rich shown to induce higher responses
[73]. Interestingly, some viruses are exquisitely sensitive to
IFN-?, casting doubts on results from early studies using
siRNA against viruses where immune responses were not
investigated. Indeed it was recently shown that modification
of siRNA to remove immune stimulation while retaining
gene silencing ability removed both in vivo and in vitro ef-
fects of an siRNA against influenza A [74]. The develop-
ment of modifications such as the addition of 2'O-methly
groups or 2'-fluoro groups to remove immune activating
ability of siRNA has allowed gene silencing effects to be
separated from immune activating effects of siRNA [46].
mental results using siRNA, the efficacy of immune activat-
ing siRNA, however, may result in improved antiviral ther-
apy compared to non immune activating siRNA. The addi-
tion of immune activation to siRNA has been shown to im-
prove siRNA potency against of influenza A, and human
papilloma virus [18, 75]. As many viruses also dampen inter-
feron-? responses to aid in their survival [76, 77], using
siRNA to reactivate these responses is likely to assist viral
eradication. Like any drug that manipulates the immune sys-
tem, great care when testing and anticipation of any impact
on patient immune system are required when testing siRNA
in humans.
While an important consideration in interpreting experi-
3. RNAi SCREENING TO IDENTIFY NOVEL ANTI-
the development of large RNAi screens and it is here that the
power of RNAi comes to the fore. It is now possible to si-
lence most genes individually within a cell using RNAi.
Various RNAi libraries, both shRNA- and siRNA-based, are
available and have been used in genome wide screens to in-
terrogate biology in an unbiased fashion. Such screens have
been performed in the presence of virus and have resulted in
the identification of many previously undiscovered genes
being uncovered that are absolutely required for replication.
Each new gene identified theoretically represents a new drug
target. We have undertaken such a screen using the kinome
RNAi subset, that is all the genes that encode for kinases, in
order to define new human papillomavirus susceptibility
genes (manuscript in preparation). Here we will review and
discuss the RNAi screening efforts to date.
A recent tool in the discovery of novel antivirals has been
3.1. Screening Technologies
cells and transfect them with an RNAi moiety before infect-
ing them with the virus under investigation. Cells that sur-
vive, e.g. are not lysed, fused or otherwise infected, must
have lost a vital gene required for virus replication. There are
two commonly used approaches for RNAi genome-wide
screening that utilise either synthetic siRNA libraries (via
The concept of the RNAi screen is rather simple, seed
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RNA Interference for Viral Infections Current Drug Targets, 2012, Vol. 13, No. 11 1415
liposome-mediated transduction) or shRNA (via viral trans-
duction). The lipid-mediated siRNA transfection is usually
performed in multiwall microtiter plates such as 96-or 384-
well plate formats, where individual genes are knocked down
in each well (Fig. 1) [78-81]. This makes identification of the
target gene simple. In contrast, the shRNA method is often
performed in a pooled format where the whole library is in-
troduced into the cells and a subsequence selection method
(usually PCR based) is then undertaken to identify target
genes [82-85]. Both methods aim to identify the crucial gene
or set of genes that are implicated in the phenotype under
investigation and exactly localize the phenotype to the fun-
damental nodes of a pathway.
Fig. (1). Illustrated diagram of siRNA vs shRNA to identify HIV
host factors required for viral replication. Figure adapted from [1].
both have been used successfully in screens. Moreover, each
method has advantages and drawbacks. For instance, in the
synthetic siRNA method, the plates can be monitored by
cell-based biological assays such as cell lysis, proliferation
assays, cytotoxicity assays and/or nucleotide incorporation
assays. A major advantage of this method is the multiplexed
high-throughput data, which can be obtained from each well.
Furthermore, the identity of genes is simply allocated by the
well ID within the microtiter plates [81, 86-92]. However,
when performing very large-scale screenings, a high number
of microtiter plates are needed, and this requires automated
platforms to enable reproducibility and avoid variability.
Additionally, the method relies on potent cell transfection by
siRNA-liposomes and therefore requires easily-transfectable
cells, a situation unfavourable for use with primary cells,
which are difficult to transfect. On the other hand, viral
shRNA can be packaged into a lentiviral vector and used to
transduce most cell lines, including primary cells, in a pooled
format. It can be stably incorporated into the genome or
Although each method is performed in a different way,
maintained under drug selection. Using this method it is fea-
sible to introduce genetic material into a broader range of
dividing and non-dividing cells over longer periods, which is
highly advantageous compared to siRNA transfection. An-
other major advantage of the shRNA method is that it can be
performed in simple laboratory settings without the need for
expensive automated platforms [1, 82, 93-97]. Increasing
RNAi investigational findings make it obvious that the RNAi
machinery within the human cells has a role in restricting
viral infection [98-101]. As a result, viruses have developed
RNAi suppressors that may act to alter the small non-coding
RNA profile in virus infected host cells [102-105].
ful screen and these will vary depending on the platform of
choice, readout assay and RNAi library chosen. In terms of
the screen itself there are three important steps (Fig. 2).
1. The primary screen. Screening of pools or single siR-
NAs/shRNAs. Often a pool of 4 siRNA/genes is used
here (e.g. Dharmacon siGENOME library). Appropriate,
multiple controls must be used including positive (eg
PLK1) and negative control siRNAs. Starting targets =
There are many factors involved in developing a success-
2. A confirmation screen. A repeat of the primary screen
target genes that appear interesting from step 1. This en-
sures inter-screen variation is controlled for. Optionally
the 4 siRNA pools can be de-convoluted here to check
for specificity. Targets = 100s
3. A Validation Screen. Re-testing of targets from 1 and 2
using new siRNAs or shRNAs targeted to new regions of
the target gene (e.g. Dharmacon ON-Targetplus). This
will eliminate off-target effects and independently con-
firm the gene as a bone fide target.
published that have identified novel host factors required for
viral replication which we review below.
Several genome-wide RNAi screens have been recently
Human Immunodeficiency Virus (HIV)
cial in the HIV lifecycle have been undertaken [1]. Two of
these studies have used different pools of siRNA transfected
into engineered HeLa cells expressing viral co-receptor CD4
followed by infection with the virus [106, 107]. Brass et al.
investigated both the early and late phases of the HIV lifecy-
cle by looking for p24 (early replication), taking supernatants
and incubating them on fresh cells with a Tat-responsive ß-
galactosidase (ß –Gal) gene to read out viral production
[107]. The screen was performed using siRNAs targeting
21,121 genes. Pools were classified a hit if the activity of the
p24 or ß-Gal was reduced by more than or equal to two stan-
dard deviations (SD) from the plate mean. Another criterion
for hit validation was that siRNA should not decrease cell
viability by more than two SD. From this, 386 pools met
these criteria and were further validated using the four-
pooled siRNA in a separated format, resulting in 273 genes
identified. Zhou et al. used a similar strategy and found 232
genes play some role in HIV replication. Interestingly, only
fifteen genes in common were found between these two stud-
ies, with seven previously known to be involved in HIV rep-
lication including CD4 and CXCR4 [106]. A third screen
Several RNAi-based screens looking at host factors cru-
Page 6
1416 Current Drug Targets, 2012, Vol. 13, No. 11
Blake et al.
identified 295 genes that also had limited overlap with each
of the previous two studies [108].
RNAi screens and may result from many factors. A meta-
analysis of these three siRNA screens attributed the lack of
overlap to experimental noise, differences in sampling time,
and variations in hit selection criteria [109]. It was calculated
that there was only a 50% chance of the top 300 top hits be-
ing obtained in an exactly repeated screen.
This lack of overlap appears to be a common theme in
lentiviral -shRNA system targeted to 54,509 transcripts was
recently undertaken using an endpoint assay of inhibition of
virus replication as a readout [110]. Of the 54,509 tran-
scripts, only 18.2% could be silenced without affecting Jur-
kat cell viability, and of these, 252 transcripts were identified
as important in HIV replication. While these hits demon-
strated little overlap with the previously mentioned studies,
there was significant overlap in the pathways these genes
belonged to, suggesting that RNAi screens do validate in this
manner and point to potentially useful areas of therapy.
Taken together, all four screening studies have identified
novel genes which may play crucial roles in HIV replication.
Of these, 40 genes were common at least in two screens out
of the four studies.
A more robust study using Jurkat human T-cells and a
ported in mammalian cells which have uncovered surprising,
previously unthought-of, host factors that appear to be essen-
tial for influenza replication. Two of these studies have in-
vestigated the early and middle phases of the influenza life-
cycle [111, 112], while the third study considered the com-
A number of genome-wide RNAi screens have been re-
plete lifecycle [113]. Karlas and colleagues used A549 cells
and identified 287 genes required for viral replication. Of
these, 119 hits were found to inhibit the H1N1 strain and 121
inhibit the influenza A virus strains originating from swine,
with 60% of these genes working against both types. For
example, they identified DNA SON as an important factor as
well as CDC-like kinase 1 (CLK1). Use of a small molecule
inhibitor against CLK1 also reduced the viral replication
significantly, showing the importance of this gene for viral
replication [113]. In another study by Konig et al., the RNAi
genome-wide screen revealed 295 cellular factors essential
for viral replication. Only 219 hits were confirmed to play
crucial roles for influenza virus growth, and subsequent
analysis of the data showed 23 genes were important in viral
entry. The use of small molecule inhibitors against several
factors inhibited viral growth, confirming the importance of
these identified target genes [111]. In addition, Brass et al.
identified 121 cellular factors which may contribute to viral
replication. For example, they found that interferon-
inducible transmembrane proteins (IFITM) 1, 2 and 3 con-
strain an early step of influenza A virus replication. In their
study, the IFITM proteins hinder the early replication of
flaviviruses such as dengue virus and West Nile virus [112].
Once again these three RNAi genome-wide screens only
had 32 genes in common.
West Nile Virus (WNV)
terized by single-stranded and positive-sense RNA. Krishnan
et al. used a siRNA-based screen to identify novel cellular
factors that are involved in viral replication. HeLa cells were
transfected with pools of siRNA and 24 hours post-
transfection cells were fixed and viral envelope was meas-
WNV is a member of the Flaviviridae, a family charac-
Fig. (2). Scheme of RNAi screening to identify novel host cell factors in viral replication.
Page 7
RNA Interference for Viral Infections Current Drug Targets, 2012, Vol. 13, No. 11 1417
ured by immunofluorescent staining. Hit selection criterion
was based on a threshold to 2-fold change in comparison to
the controls. Based on this criterion, 305 genes that affect
WNV were identified. Of these, 283 genes, when silenced,
severely decreased viral gene expression, suggesting that
these genes play crucial roles in viral replication. Of these
genes, vacuolar ATPase was recognized as a viral replication
enhancer [114, 115].
Dengue Virus (DENV)
of infection to mankind throughout the globe. Using a ge-
nome-wide RNAi screen, Sessions et al. screened 22,632
genes in Drosophila melanogaster cells. Only 116 host fac-
tors were identified from the screen. Some of these dengue
virus host factors (DVHF) had previously been identified as
important, such as vacuolar ATPase and ?-glucosidases, al-
though most of the newly identified DVHF were novel. Ses-
sions et al. identified DVHF which were essential for viral
replication in human cells, and found that of the original 116
mosquito genes, only 82 were critical in human cells [116,
Dengue is an arthropod-borne viral disease with high risk
Hepatitis C Virus (HCV)
preventive vaccine against HCV and much hope has been
placed in genome-wide RNAi screens to identify host factors
that are essential for HCV lifecycle. Tai et al. identified 96
cellular host factor proteins that are required for HCV repli-
cation, with a remarkable number of these factors being im-
plicated in vesicle organization and biogenesis [118, 119].
Among the identified genes, phosphatidylinositol 4-kinase
(PI4KA) and COPI vesicle coat complex were tested against
some selective small molecule inhibitors. The outcome re-
vealed the inhibition of HCV growth confirming the impor-
tance of these host factors for viral replication.
To date, there is a lack of selective antiviral therapy or
Human Papillomavirus (HPV)
targeting HPV. HPV is a double-stranded small DNA virus.
The virus is subcategorized into two main serotypes, high-
risk HPV (HR-HPV) and low-risk HPV (LR-HPV). The HR-
HPV is responsible for approximately 99.7% of cervical can-
cer cases among women. In our laboratory an siRNA-based
screen was carried out on 779 genes from the kinome library
on CaSki cells (HPV-16 positive cervical cancer cell line),
C33A cells (cervical cancer cell line negative for HPV), and
HaCaT cells (spontaneous, immortalized human keratino-
cytes). Four different cell-based biological assays including
cell metabolism, cell cytotoxicity, nucleotide incorporation
assay and cell count were used as an endpoint assay for hit
selection. Of the 779 kinases, only 55 genes demonstrated
lethal effect to CaSki when silenced, with minor effect on
C33A and HaCaT. A second validation screen was per-
formed to validate these target genes. In the validation
screen, two additional cervical cancer cell lines, HeLa (HPV-
18 positive) and SiHa (HPV-16 positive) were incorporated.
Eight genes showed lethal effects against cervical cancer cell
lines with minor effect on HaCaT. Using pharmacological
inhibitors, one of these eight genes affected CaSki and HeLa
Here at the AID we have undertaken an RNAI screen
cells' growth in comparison to primary fibroblasts. Papers
describing these genes are currently under preparation.
to identify genes that suppress expression from HPV18-long
control region (LCR) [120] Examining more than 21,000
genes, the primary screen has confirmed only 96 cellular
genes that are essential in the suppression of the HPV18-
LCR. Most of these genes primarily work together with the
E2 whereas some other act independently to repress the
LCR. The secondary screening experiments have confirmed
the cellular demethylase JARID1C/SMCX and EP4000.
More importantly, the authors noted that, their findings sup-
port their hypothesis that E2 engage different cellular path-
ways to down-regulate the expression of the oncogenic ac-
tivities of the HPV18.
In addition, Smith et al. undertook a genome-wide screen
4. CONCLUSIONS OF siRNA THERAPY FOR VI-
clinical tool, early difficulties in delivery have prevented it
from reaching its potential. However, with the results of sev-
eral clinical trials that supposedly demonstrate its effective-
ness resulting in less than optimal outcomes, the field awaits
true success. Most issues centre around solving the delivery
problem. Genome-wide RNAi screening has identified a
whole new range of host cell factors currently under investi-
gation. While the screen process itself needs to be carefully
worked out and controlled, results are promising, with many
new targets already having small molecule inhibitors ready
to use in the clinic.
While siRNA has been long proposed as an excellent
flicts of interest.
The authors confirm that this article content has no con-
Declared none.
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de Vries W, Berkhout B. RNAi suppressors encoded by pathogenic
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Haasnoot J, de Vries W, Geutjes EJ, Prins M, de Haan P, Berkhout
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Received: January 30, 2011 Revised: November 08, 2011 Accepted: May 18, 2012


text:
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Matt Vensel is in his first year at the Star Tribune after covering the Ravens for the Baltimore Sun for six years. He is a Pittsburgh native and a Penn State grad. Follow him at @mattvensel.
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EAST RUTHERFORD, N.J. -- Vikings left tackle Matt Kalil will play Monday night against the New York Giants after being listed as questionable because of back tightness.
Kalil missed two days of practice last week after experiencing some tightness in his back. He told reporters on Saturday that he had made progress and was optimistic he would be able to play.
The most notable inactive was Matt Cassel, who went from starting quarterback the previous two games to No. 3 on the depth chart this week, behind Josh Freeman and Christian Ponder.
The other inactives for the Vikings are cornerback A.J. Jefferson, wide receiver Rodney Smith, linebacker Gerald Hodges, offensive guard Jeff Baca, defensive tackle Chase Baker and defensive end Justin Trattou.
For the Giants, running back Brandon Jacobs is inactive because of a hamstring injury. The Giants' second-leading rusher, David Wilson, also is out because of a neck injury. That leaves veteran Peyton Hillis, who signed with the Giants this past week.
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Tag: cap
Apple, Inc. market cap now exceeds Walmart
Apple and Walmart have been playing stock market tag over the past few weeks to see which company's market capitalization would be higher. At the time of this writing, Apple's market capitalization of $213.98 billion exceeds Walmart's market cap of $211.14 billion -- a difference of $2.84 billion....
Goofy iPod Accessory: iSoundCap
I guess the $2 billion iPod accessory market is inspiring everyone to snag a piece of the pie, no matter how... unique their accessory concept might be. Behold: the iSoundCap, a 'breakthrough' way to carry your iPod nano or shuffle. As you can see from the diagram, this stylish cap comes with a...
1000 song hack for iTunes phones?
For those out there who find the 100 song cap (amongst other things) on iTunes phone a deal breaker, Engadget Mobile might have found your saving grace. A forum at MotoModders has the details and simple steps you can take to run a simple hack on any iTunes phone (except the v3i) to raise the...
iTunes phone upgrades only for non-U.S. phones with < 100 song limit
I hope no one ran out and bought a ROKR or slick new SLVR after last night's post about the ability to upgrade iTunes phones. Turns out it was more or less a false alarm - at least for those who already have a phone with a 100 song limit. Eddie Cue, iTunes' product manager, got in touch with...
Apple removing the iTunes phone 100 song cap?
I can neither confirm nor deny the legitimacy of this, but TUAW reader Jake sent us a tip with an iTMS screenshot detailing an upcoming possibility of removing the 100 song cap on iTunes phones. From the screenshot it looks like Apple might soon offer the ability to purchase what I would assume to...
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Books & Periodicals > Catalogues of Music Collections in Bavaria (KBM)
Stadtpfarrkirche St. Jakobus und Tiburtius in Straubing
Band 14, Bischöfliche Zentralbibliothek Regensburg, 5
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Musicians throughout the world are familiar with and treasure our blue Urtext editions – top quality editions of great classical compositions. Urtext and Henle are synonymous. The musician is offered a musical text which solely reflects the composer's intentions.


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WATCH: Trailer For 'Minions,' The 'Despicable Me' Spinoff
We had no idea they were making a Despicable Me spinoff, but apparently they are. It's called 'Minions' — watch the trailer here.
Just How Many Refunds Have Been Issued From Chicago's Red Light Cameras?
Remember how the Chicago Tribune reported that were some unusual spikes in the number of tickets issued by some red light cameras?
Who's Ahead In The Latest Polls For Illinois Governor?
It seems like there's a different poll, with a different result every week.
President Obama Could Mess Up Chicago Traffic Day!
President Obama will be in town later today and that means there is always a potential for some major traffic problems because of it.
What's The Latest Mud Slinging Going On Between Bruce Rauner & Governor Quinn?
It seems like every day Governor Quinn and his Republican opponent Bruce Rauner find something new to rip into each other about!
'Turntable Tuesday' Video: 'Dancing With Myself' By Billy Idol
Did did you know that "Dancing With Myself" was actually released twice?
Did You Get As Much As Jesse Jackson Jr. When You Refinanced Your Home?
Jesse Jackson Junior is still in prison and will likely still be there for awhile.
How Can Chicago Crime Be Up AND Down?
Here's one of those stats that leaves you scratching your head.
Jimmy Page 'Fed Up' With Robert Plant's Games, Wants To Tour Again With Led Zeppelin
Jimmy Page makes it absolutely clear that he'd love to tour again, but there's just one holdup…
This CTA Bus Driver Almost Got The Ax This Weekend!
Thankfully no one was hurt, but did you hear what happened on a CTA bus this past weekend!


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I'll e-mail you a copy of the history of the Ragan family with William E. Ragans mother and fathers information. William and his 2nd wife Mary are supposedly buried in Guion, Izard Co., AR. Within 20 miles from where I live. I have yet been able to find their gravesite.
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Guilds look to be getting a decent chunk of attention too. Once you've reached level 40 (of 80) you'll be able to start doing some serious crafting. But rather than just churning out attractive hats, this will allow you and your guildmates to establish a large, instanced base for celebrating your glory, with as many as 150 players sharing the space.
It's an amusing goal to aim for, and there's a small tactical advantage too, as the area offers opportunities for resource gathering, which will come in useful for PvP action.
Then dozens more details present themselves. Over 100 voice actors have been used for the NPCs, and from our brief play, they're pretty decent. It's a hefty relief to hear a Scottish accent in a game performed by an actor who's at least visited the country, even more so one who can offer an authoritative and impressive brogue.
There's a day/night cycle, which affects the behaviour of the NPCs. There are 2,500 animations for male and female characters, with hundreds of emotes to match (including getting drunk and the subsequent peeing).
What remains to be seen is whether the many years of effort will connect into a coherent and engrossing MMO. The combat, while much improved, will still provide an obstacle to new players merely by dint of being different from World of Warcraft.
We'd really like to see Conan succeed - hell, we'd really like to see any MMO different enough from WoW succeed. The open beta starts any moment now.


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Effects of abscisic acid (ABA) on grain filling processes in wheat
- A. Ahmadi,
- D.A. Baker
- … show all 2 hide
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The effect of in situ water stress on the endogenous abscisic acid (ABA) content of the endosperm and the in vitro application of ABA on some important yield regulating processes in wheat have been studied. Water stress resulted in a marked increase in the ABA content of the endosperm at the time close to cessation of growth. Application of ABA to the culture medium of detached ears reduced grain weight. Exogenously applied ABA, at the highest concentration (0.1 mM) reduced transport of sucrose into the grains and lowered the starch synthesis ability of intact grains. In vitro sucrose uptake and conversion by isolated grains was stimulated by low ABA concentrations (0.001 mM) in the medium but was inhibited by higher concentrations. ABA application had no effect on sucrose synthase (SS) and uridine diphosphate glucose pyrophosphorylase (UDP-Gppase) activities, whereas adenosine diphosphate glucose pyrophosphorylase (ADP-Gppase), soluble starch synthase (SSS), and granule-bound starch synthase (GBSS) activities were reduced. These results raise the possibility that water stress-induced elevated levels of endogenous ABA contribute to reduced grain growth.
- Barlow EWR, Donovan GR and Lee JW (1983) Water relations and composition of wheat ears grown in liquid culture: Effect of carbon and nitrogen. Aust J Plant Physiol 10: 99–108
- Bensen RJ, Boyer JS and Mullet JE (1988) Water-deficitinduced changes in abscisic acid, growth, polysomes, and translatable RNA in soybean hypocotyls. Plant Physiol 88: 289–294
- Bewley JD and Black M (1994) Seeds. Physiology of Development and germination, Second Edition. New York and London: Plenum Press
- Borkovec V and Prochazka S (1992) Pre-anthesis interaction of cytokinins and ABA in the transport of 14C-sucrose to the ear of winter wheat (Triticum aestivum L.). J Agr Crop Sci 169: 229–235
- Bray EA (1991) Regulation of gene expression by endogenous ABA during drought stress. In: Davies, WJ and Jones HG (eds) Abscisic Acid. Physiology and Biochemistry. BIOS Scientific Publishers, pp 81–98
- Brenner ML, Brun WA, Schussler J and Cheikh N (1986) Effects of endogenous and exogenous plant growth substances on development and yield of soybeans. In: Bopp M (ed) Plant Growth Substances. Berlin Heidelberg: Springer-Verlag, pp 380–386
- Caley CY, Duffus CM and Jeffcoat B (1990) Effects of elevated temperature and reduced water uptake on enzymes of starch synthesis in developing wheat grains. Aust J Plant Physiol 17: 431–439
- Cheikh N and Brenner ML (1992) Regulation of key enzymes of sucrose biosynthesis in soybean leaves. Effect of dark and light conditions and role of gibberellins and abscisic acid. Plant Physiol 100: 1230–1237
- Chraibi A, Palms B, Druart N, Goupil P, Gojon A and Ranbour S (1995) Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells. J Exp Bot 46: 1525–1533
- Dembinska O, Lalonde S and Saini HS (1992) Evidence against the regulation of grain set by spikelet abscisic acid levels in water-stressed wheat. Plant Physiol 100: 1599–1602
- Doehlert DC (1993) Sink strength: Dynamic with source strength. Plant Cell Env 16: 1027–1028
- Donovan GR and Lee JW (1977) The growth of detached wheat heads in liquid culture. Plant Sci Let 9: 107–113
- Felker FC, Peterson DM and Nelson OE (1984) [14C] sucrose uptake and labeling of starch in developing grains of normal and segl barley. Plant Physiol 74: 43–46
- Goldbach H and Goldbach E (1977) Abscisic acid translocation and influence of water stress on grain abscisic acid content. J Exp Bot 28: 1342–1350
- Haeder HE and Beringer H (1981) Influence of potassium nutrition and water stress on the content of abscisic acid in grains and flag leaves of wheat during grain development. J Sci Food Agric 32: 552–556
- Hawker JS and Jenner CF (1993) High temperature affects the activity of enzymes in the committed pathway of starch synthesis in developing wheat endosperm. Aust J Plant Physiol 20: 197–209
- Ho LC (1988) Metabolism and compartmentation of imported sugars in sink organs in relation to sink strength. Ann Rev Plant Physiol Plant Mol Biol 39: 355–378
- Kermode AR, Oishi MY and Bewley JD (1989) Regulatory roles for desiccation and abscisic acid in seed development: A comparison of the evidence from whole seeds and isolated embryos. In: Stanwood PC and McDonald MB (eds) Seed Moisture. CSSA Special Publication, pp 23–50
- King RW(1976) Abscisic acid in developing wheat grains and its relation to grain growth and metabolism. Planta 132: 43–51
- King RW (1982) Abscisic acid in seed development. In: Khan AA (ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. Elsevier Biomedical Press, pp 157–181
- Lu J-L, Ertl JR and Chen C-M (1992) Transcriptional regulation of nitrate reductase mRNA levels by cytokinin-abscisic acid interactions in etiolated barley leaves. Plant Physiol 98: 1255–1260
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- Myers PN, Setter TL, Madison JT and Thompson JF (1990) Abscisic acid inhibition of endosperm cell division in cultured maize kernels. Plant Physiol 94: 1330–1336
- Ober ES, Setter TL, Madison JT, Thompson JF and Shapiro PS (1991) Influence of water deficit onmaize endosperm development. Enzyme activities and RNA transcriptions of starch and zein synthesis, abscisic acid, and cell division. Plant Physiol 97: 154–164
- Ober ES and Setter TL (1990) Timing of kernel development in water stressed maize: Water potentials and abscisic acid concentrations. Ann Bot 66: 665–672
- Quarrie SA (1991) Implications of genetic differences in ABA accumulation for crop production. In: Davies WJ and Jones HG (eds) Abscisic Acid. Physiology and Biochemistry. BIOS Scientific Publishers, pp 227–244
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- Sowokinos JR and Varns JL (1992) Induction of sucrose synthase in potato tissue-culture. Effect of carbon source and metabolic regulators on sink strength. J Plant Physiol 139: 672–679
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- Title
- Effects of abscisic acid (ABA) on grain filling processes in wheat
- Journal
Plant Growth Regulation
Volume 28, Issue 3 , pp 187-197
- Cover Date
- 1999-07-01
- Print ISSN
- 0167-6903
- Online ISSN
- 1573-5087
- Publisher
- Kluwer Academic Publishers
- Additional Links
- Topics
- Keywords
- grain filling
- water stress
- wheat
- Industry Sectors
- Authors
- A. Ahmadi (1)
- D.A. Baker (1)
- Author Affiliations
- 1. Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent, TN25 5AH, U.K


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Jessee, GPCA executive director, has been with the association for 27 years.
NORCROSS, GA – In a surprise announcement at the Georgia Pest Control Association Conference in Athens, Ga., Valera Jessee, GPCA Executive Director, was elected into the GPCA Hall of Fame.
Jessee joined the GPCA staff 27 years ago and has worked tirelessly with 27 different presidents, boards and committees. "Her greatest talent," according to Jeffrey Bodine Sinyard, "is her ability to work with different personalities and styles in a friendly, pleasant manner to achieve mutual goals."
The Hall of Fame award (previously known as honorary members of GPCA) acknowledges the work that an individual has done to grow the Georgia pest control industry and GPCA. Jessee is the first to be elected to the newly named Hall of Fame. Eunice Hall of Dow talks about Jessee's worthiness for this award. "There has been no one more deserving than Valera to receive this honor and I consider her not only a colleague but a good friend. Her contributions to GPCA and the pest control industry in Georgia are unmatched." Coworker Connie Rogers also attributes the success of GPCA to Jessee's professionalism and integrity.
Chuck Carney, Cook's Pest Control said, "The recognition of Hall of Fame Member for a lady who has given so much of herself to the Georgia Pest Control Association is just a small way to say thank you for what she has done and continues to do. We all wanted to thank Valera with this special recognition."
A new study from Penn Medicine researchers in the Center for Clinical Epidemiology and Biostatistics demonstrated that bed bugs, like the triatomines, can transmit Trypanosoma cruzi, the parasite that causes Chagas disease.
The National Pest Management Association (NPMA) is seeking a qualified regulatory affairs professional to direct the day-to-day management and execution of NPMA's federal and state regulatory affairs programs
The company's latest Technical Bulletin provides an in-depth description of variables to consider when developing integrated pest management (IPM) programs in school settings.
The company has been selected by the National Park Service to install and maintain effective termite control systems for 14 national historic sites in the Delaware Valley, including Independence Hall.
Ronnie Caldwell, president of Innovative Pest Management, Denver, N.C., is one of the stars of the new Bruce Hedrick video 'A Better Man.'


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The median value of homes changing hands in Manteca ended 2.5 years of freefall sometime during the summer of 2009.
Is spending $89,000 on a lobbyist in Washington, D.C., worth it?
Juveniles arrested for burglary in Manteca may soon be forced to wear ankle monitors.
The Class of 2010 at both Ripon High and Ripon Christian High will soon receive their diplomas.
Dale Gray called the triple-attraction combination a win-win for everyone concerned.
It's one thing to try and imitate something historic.
to rest on May 10, 2010 in Modesto, California. She was born on October 29, 1928.She has resided in Modesto, Ca for over 6 years and previously of Manteca. Amalia joins her husband in heaven Jose C. Valdez and their daughter Martha Valdez.
San Joaquin County Office of Education named six exemplary school employees as the county's 2010 Classified School Employees of the Year.
The Manteca Federated Women's Club recently announced the 2010 scholarship recipients.
Three different sizes of car seats were installed in parents' vehicles throughout much of the day Saturday at a CHP inspection station that was set up in the Best Buy parking lot adjacent to the Highway 120 Bypass at The Promenade Shops at Orchard Valley.
An $8 million "state of the art" cancer center is on the horizon for Manteca under the name of Valley Cancer Center, Inc.
Young kids in his neighborhood call Austen Nunes a show-off.
Don't look for a wider bridge deck any time soon at the Airport Way and Highway 120 Bypass interchange.
The game has come a long way since it was first born in a barn in the Pacific Northwest and named after a pesky dog that kept taking the ball from the middle of the game.
The double-crested cormorant is not on any endangered species list.
It is going to cost nearly $150 million for levee upgrades to protect Manteca, Lathrop, Weston Ranch, and parts of rural south Manteca from a 200-year flood.
It's now the $310 million question.
Jim Anderson - a popular home-grown rock singer that pays tribute to Elvis Presley's early years - is performing on the Main Stage of the 30th annual Sunrise Kiwanis Pumpkin Fair this weekend in downtown Manteca.
The Moving Wall - an 80 percent replica of the Vietnam Memorial in Washington, D.C., - is returning to Manteca a little more than 20 years after its first appearance.
Editor's note: This is an abbreviated version of a column about the Manteca High Vietnam Moving Wall ceremonies that former Manteca High student and Congressional Medal of Honor recipient Sammy Davis appeared at that was published in March of 1993.
Good luck getting California's diverse special interest groups to agree on anything.
Educator Shane Smith is on the ballot for the Nov. 4 vote for Ripon City Council election even though he says he has been talked out of running because he is relatively new to the community.
The Home Energy Opportunity (HERO) program is considered as one of the fastest growing energy efficiency financing solution in the country.
You can tell a lot about a city by its garbage.
"Fight Hunger, Spark Change" is the theme of the current online voting contest that could give the San Joaquin and Stanislaus Counties Food Bank in Manteca a $60,000 prize courtesy of Walmart.
Manteca is rallying to help 19-year-old Jose Maltez.


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Originally written on Nationals Enquirer | Last updated 11/16/14
So Bryce Harper has accepted an invitation to participate in the Home Run Derby at the All-Star game next week. Raise your hand if you're worried that he'll hurt himself somehow and/or mess up his swing forever! Oh well, his brother Bryan doesn't seem worried: Can't wait to watch Bryce in the HR Derby!! Gonna put on a show for the fans in NYC!! @Nationals @Bharper3407 — Bryan Harper (@BryanHarper45) July 8, 2013 Harper joins Captain David Wright, Michael Cuddyer and Carlos Gonzalez for the NL. They'll face Captain Robinson Cano, Chris Davis, Prince Fielder, and a player to be named later for the AL. (Screencap via CSNWashington.com video) Befriend Nats Enquirer on Facebook. Follow on Twitter. Related articles Bryce Harper on his "play me or trade me" text to Davey: "I was pretty serious about what I said." Harrisburg Senators gave away Bryce Harper pillowcases on Saturday Davey Johnson will rest Bryce Harper for the r...
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A Foreign Policy that fails to factor the foreign.
October 7, 2013 by 8 Comments
Raymond Ibrahim is a Shillman Fellow at the David Horowitz Freedom Center, a Judith Friedman Rosen Writing Fellow at the Middle East Forum and a CBN News contributor. He is the author of Crucified Again: Exposing Islam's New War on Christians (2013) and The Al Qaeda Reader (2007).
Debunking Anti-Anti-Islamist Myths
January 14, 2010 by 10 Comments


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Baby Lyrics - Gotye
Live from day to the next day
Try to satisfy the cravings before they
Consume you
Colours are brighter
The world can excite you
When you're under its spell
You can tell
All too well
That without it life would be hell
And time is passing you by
It marks no moment of your life
When you submit yourself to be numb,
The fear disappears,
And everything seems clear
When the emptiness is upon you
It fills you up like a balloon
And you float
High up over the real world,
And think of when
You were a little girl again
Where would you go, baby?
What could you do?
Didn't you hear, baby?
Nothing is sure but uncertainty
You can feel the hunger inside you
But the longing will no longer guide you onwards,
Sinking into the floorboards
Where the rats eat at your will
And do it until,
Your belly aches
And your soul will not be still
Wrench yourself from your demons
For far too long you've believed them
When they told you you were weak
Take a step in the tunnel
With the light 'round the bend
In the end
You can start to make amends
Who knows where you'll go, baby
So much you could do!
Haven't you heard, baby?
All will be well
If you want it to
Click here to submit the Corrections of Baby Lyrics
Please Click Here to Print Baby Lyrics
The following area is only for review, if you want to submit the lyrics or the corrections of the lyrics, please click the link at the end of Baby Lyrics.


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