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Ebola therapy breakthrough

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Updated 11/23/2015

Giant Molecules Inhibit Ebola Infection

Nov 11, 2015   http://www.technologynetworks.com/medchem/news.aspx?ID=185080

European researchers have designed a “giant” molecule formed by thirteen fullerenes covered by carbohydrates which, by blocking this receptor, are able to inhibit the cell infection by an artificial ebola virus model.

 

Different studies have demonstrated that the ebola virus infection process starts when the virus reaches the cellular DC-SIGN receptor to infect the dendritic cells (of the immune system).

In this study researchers from the Universidad Complutense de Madrid/IMDEA-Nanociencia, the Instituto de Investigación Sanitaria Hospital 12 de Octubre (Madrid), and the Instituto de Investigaciones Químicas del CSIC-Universidad de Sevilla have collaborated, together with three european research groups (CNRS/Université de Strasbourg, France and Université de Namur, Belgium).

“Fullerenes are hollow cages exclusively formed by carbon atoms”, explains Nazario Martín, Professor of Organic Chemistry in the UCM and main author of the study. In this work, scientists have employed C60 fullerene, which is formed by 60 carbon atoms and has the shape of a truncated icosahedron, which resembles a football ball.

102815_web.jpg

These molecules decorated with specific carbohydrates (sugars) present affinity by the receptor used as an entry point to infect the cell and act blocking it, thus inhibiting the infection.

Researchers employed an artificial ebola virus by expressing one of its proteins, envelope protein GP1, responsible of its entry in the cells. In a model in vitro, this protein is covering a false virus, which is able of cell infection but not of replication.

“We have employed a cell model previously described in our lab which consists in a cell line of human lymphocytes expressing DC-SIGN receptor, which facilitates the entry of the virus in Dendritic Cells”, points out Rafael Delgado, researcher of the Hospital 12 de Octubre, and other of the authors of the study.

By blocking this receptor and inhibiting the virus infection, the authors think that the dissemination of the virus would decrease and the immune response increase, but this idea has still to be developed with in vivo studies.

The biggest fullerene system in the lab

The system designed by the chemists, based on carbon nanostructures developed in the UCM, mimic the presentation of carbohydrates surrounding virus like ebola or VIH.

The team has achieved an unprecedented success in fullerene chemistry and dendritic growth: connecting in one synthetic step twelve fullerene units, each with ten sugars, to other central fullerene, creating a globular superstructure with 120 sugar moieties on its surface, “this is the fastest dendrimeric growth developed in a laboratory up to now” says Beatriz Illescas, Professor in the UCM and coauthor of the work.

According to scientists, the results highlight the potential of these giant molecules as antiviral agents. “This work open the door to the design and preparation of new systems to inhibit the pathogens infection in cases where the current therapies are not effective or are inexistent, as occurs with the ebola virus”, clarifies Martín.

After these experiments on the cellular level, researchers will study the behavior of these systems in animal models, starting with mice. “We will study, on the one hand, the pharmacokinetics and, on the other, the antiviral activity in vivo” explains Javier Rojo, researcher of the Instituto de Investigaciones Químicas del CSIC and other of the authors of the study. Once the most effective compound has been identified, studies using the true ebola virus could be carried out.

 

 

http://www.sciencedirect.com/science/article/pii/S0092867400806935

DCSIGN, which is abundantly expressed by DC both in vitro and in vivo, … Whereas ICAM-3 binding by monocytes is for the greater part LFA-1 … The specificity of this adhesion receptor on DC for ICAM-3 is demonstrated by the ….

 

http://www.bloodjournal.org/content/100/5/1780.full.pdf

This subset coexpresses CD14, CD16, and CD33 and is thus of myeloid origin. In contrast to. CD14 monocytes, DCSIGN blood cells.

 

http://www.jimmunol.org/content/168/5/2118.full

Mar 1, 2002 Several receptors expressed by immature DCs belong to the C-type lectin superfamily, … Here, DCSIGN efficiently transmits the virus to T lymphocytes

 

http://journals.plos.org/plospathogens/article%3Fid%3D10.1371/journal.ppat.0020070

Jul 14, 2006 Although B cells that express DCSIGN do not replicate HIV-1, they serve as … receptors [12–15], with conflicting reports on expression of DCSIGN[16,17]. …..
human herpesvirus 8 infects DC and macrophages via DCSIGN …

 

http://www.jci.org/articles/view/25105/files/pdf

Results. The effect of human milk on direct HIV-1 infection of CD4+ T lymphocytesexpressing the DCSIGN receptor (Raji-DCSIGN) (8).

 

 

An indictment of Ebola response  

Panel calls for reform of global public health system in wake of epidemic

By B. D. Colen, Harvard Staff Writer

http://news.harvard.edu/gazette/story/2015/11/an-indictment-of-ebola-response/

 

http://media.news.harvard.edu/gazette/wp-content/uploads/2015/11/110515_Ebola_020_605.jpg

“The most egregious failure was by WHO in the delay in sounding the alarm,” said Harvard’s Ashish Jha.

An independent group of 19 international experts, convened by theHarvard Global Health Institute and the London School of Hygiene and Tropical Medicine (LSHTM), today issued a scathing analysis of the global response to the 2014-15 Ebola outbreak in West Africa.

The members of the Harvard-LSHTM Independent Panel on the Global Response to Ebola said that while the 2014-15 Ebola outbreak “engendered acts of understanding, courage, and solidarity,” it also caused “immense human suffering, fear and chaos, largely unchecked by high-level political leadership or reliable and rapid institutional responses.”

The report, published in the prestigious British medical journal The Lancet, is especially hard on the World Health Organization (WHO), which the panel contends failed to provide the leadership and support needed to deal properly with the outbreak of hemorrhagic fever that infected more than 28,000 people and claimed more than 11,000 lives.

The authors of the report, who were affiliated with, but functioned independently from, such disparate organizations as the Council on Foreign Relations, Médecins Sans Frontières, Indiana University law school, and theAIDS Health Care Foundation, reminded readers that the Ebola epidemic “brought national health systems to their knees, rolled back hard-won social and economic gains in a region recovering from civil wars, sparked worldwide panic, and cost at least several billion dollars in short-term control efforts and economic losses.”

“The most egregious failure was by WHO in the delay in sounding the alarm,” said Ashish Jha, director of the Harvard Global Health Institute, K.T. Li Professor of International Health at the Harvard T.H. Chan School of Public Health, and a professor of medicine at Harvard Medical School. “People at WHO were aware that there was an Ebola outbreak that was getting out of control by spring … and it took until August to declare a public health emergency … Those were precious months,” said Jha.

The panel was co-chaired by Professor Peter Piot, director of the LSHTM and co-discoverer of the Ebola virus. Piot said, “We need to strengthen core capacities in all countries to detect, report, and respond rapidly to small outbreaks, in order to prevent them from becoming large-scale emergencies. Major reform of national and global systems to respond to epidemics are not only feasible, but also essential so that we do not witness such depths of suffering, death, and social and economic havoc in future epidemics. The AIDS pandemic put global health on the world’s agenda. The Ebola crisis in West Africa should now be an equal game-changer for how the world prevents and responds to epidemics.”

Liberian Mosoka Fallah of Action Contre la Faim International and a member of the panel said, “The human misery and deaths from the Ebola epidemic in West Africa demand a team of independent thinkers to serve as a mirror of reflection on how and why the global response to the greatest Ebola calamity in human history was late, feeble, and uncoordinated. The threat of infectious disease anywhere is the threat of infectious disease everywhere. The world has become one big village.”

The global response to Ebola is being examined by a number of different panels, Jha said, including a group at WHO and another at the United Nations. During the height of the epidemic in fall, 2014, Jha met with Julio Frenk, then the dean of the Harvard Chan School, and Suerie Moon, research director and co-chair of the Harvard Kennedy School’s Forum on Global Governance for Health, and a Harvard Chan faculty member. Together, they “decided this deserves independent examination; we can’t let this happen again,” Jha said.

“The Ebola outbreak is a stark reminder of the fragility of health security in an interdependent word,” the report reads, “and of the importance of building a more robust global system to protect all people from such risks.

“A more humane, competent, and timely response to future outbreaks requires greater willingness to assist affected populations, and systematic investments to enable the global community to perform four key functions: strengthen core capacities within and among countries to prevent, detect, and respond to outbreaks when and where they occur; mobilize faster and more effective external assistance when countries are unable to prevent an outbreak from turning into a crisis alone; rapidly produce and widely share relevant knowledge, from community mobilization strategies to protective measures for health workers, from rapid diagnostic tools to vaccines; [and] provide stewardship over the whole system, entailing strong leadership, coordination, priority setting, and robust accountability from all involved actors.”

Though it pulls no punches in its criticism of the ways institutions and nations responded to the Ebola crisis, the Harvard-LSHTM report is also a positive document, offering 10 concrete recommendations to strengthen public health systems and future responses.

Those recommendations fall into four areas: preventing major disease outbreaks; responding to outbreaks; producing and sharing data, knowledge, and technologies; and improving the governance of the global health system, “with a focus on the World Health Organization.”

One recommendation is that WHO create a dedicated center “for outbreak response, with strong technical capacity, protected budget, and clear lines of accountability,” and that that center be governed by a separate board independent of the WHO bureaucracy.

“Our primary goal is to convince the high-level political leaders, north and south, to seize the moment to make necessary and enduring changes to better prepare for future outbreaks, while memories of the human health costs of inaction remain vivid and fresh,” the report said.

“There is a high risk here of not learning our lessons,” said Jha. “We’ve had outbreaks like this before, and often you get thoughtful reviews, and august bodies that look at it, and people say, ‘We will get to this right away,’ and then other things draw our attention. I think we owe it to the more than 11,000 people who died in West Africa to see that that doesn’t happen this time.”

 

The Lancet 2015

http://www.thelancet.com/campaigns/ebola

Ebola—lessons learned: Authors from Harvard’s Global Health Institute and the London School of Hygiene and Tropical Medicine outline 10 proposals to help prevent future health catastrophes, based on experiences from the 2014-15 Ebola outbreak in west Africa.

Timeline infographic

Illustration demonstrating pathogenesis of vascular leak in Ebola virus disease - Copyright: Elsevier
http://www.thelancet.com/pb/assets/raw/pb/assets/raw/lancet/campaigns/ebola/ebola-main-281114.jpg

The current outbreak of Ebola in west Africa is both a public health emergency of international concern and a human tragedy.

The Lancet Ebola Resource Centre contains all related resources from The Lancet family of journals offered with free access to assist health workers and researchers in their important work to bring this outbreak to a close a quickly as possible.

Find out more about Ebola in The Lancet’s Seminar.

 

WORLD REPORT
Expert panel slams WHO’s poor showing against Ebola
John Maurice
The Lancet, July 13, 2015;Vol. 386, No. 9990, e1

Criticism of WHO’s response to the west African Ebola crisis spawned an expert review that this week proposed several solutions to restore the agency’s performance. John Maurice reports.

WHO suffers from an incapacity “to deliver a full emergency public health response” against a severe epidemic. So concluded a panel of six international health experts in a damning report released on July 7. They prescribed 21 actions aimed at restoring WHO’s “pre-eminence as the guardian of global public health”.

The panel was commissioned by WHO Director-General Margaret Chan in response to widespread criticism that WHO had mishandled its response to the west African Ebola epidemic. The panel corroborated many of the criticisms. Chief among them was the “unjustifiable” time it took WHO to declare the outbreak a “public health emergency of international concern”. Chan made this declaration 5 months after the escalating spread of Ebola had become evident. WHO officials claim that the delay in making the official declaration did not affect its operations involving some 100 staff in the field in the early months of the epidemic.

WHO’s Member States also drew sharp criticism from the panel. Many applied travel bans during the epidemic without WHO authorisation, thereby contravening the International Health Regulations (IHR) and “causing negative political, economic and social consequences for the affected countries”. Perhaps the most damning criticism of WHO came from Médecins sans Frontières (MSF), whose teams were among the first to arrive at the scene of the outbreak in March, 2014. An MSF reportpublished in March, 2015, describes how MSF was unable to convince WHO that the epidemic was out of control. “WHO officials”, the report notes, “called us alarmists”.

Four of the panel’s recommendations stand out: countries should be given incentives to comply with the IHR and disincentives, such as sanctions, when they flout them; a brand-new WHO Centre for Emergency Preparedness and Response should be created; a contingency fund of US$100 million to be used solely to finance outbreak responses should be established; and an intermediate trigger should be set up to alert the health community to a health crisis before it becomes an emergency.

Asked whether the panel’s report meets her concerns, MSF president Joanne Liu tells The Lancet: “It has many strong points for us. But how they will translate into real action on the ground” is unclear. Liu is particularly pleased with the panel’s call for greater community engagement in epidemic response efforts. “As regards an intermediate alert”, she says, “it should be based on the needs of the affected communities, not just on a perceived security risk for other countries. MSF didn’t wait for an official declaration before going into the field.”

David Heymann, head and senior fellow at the Centre on Global Health Security in Chatham House, London, wonders whether the panel’s recommendations for fundamental changes in the decision-making processes can be implemented. “WHO has a flawed structure and I’m not sure its Member States have the will to change that.” He commends the panel’s call for strengthening existing emergency response mechanisms, such as the Global Outbreak Alert and Response Network (GOARN). “This is an agile, sustainable network of epidemiologists, logisticians, and other field-support experts from WHO Member States. It goes immediately into action to prevent outbreaks from becoming emergencies of international concern and has worked extremely well in previous Ebola outbreaks and in the 2003 SARS epidemic.” He believes that the existence of GOARN, with an added external advisory group, obviates the need for the new WHO emergency response centre proposed by the panel.

Will WHO implement the recommendations? “If it doesn’t implement them now”, says Jeremy Farrar, director of the Wellcome Trust, “it will never do so, because the Ebola epidemic has really shocked people and has exposed the structural weaknesses in WHO. Reforming its emergency response capabilities means reducing the bureaucracy and speeding up its capacity to respond. And that means appointing the very best people.” Farrar is enthusiastic about the proposed creation of a new WHO emergency response body. “It should be overseen by an independent board and needs to be outside the influence of politics and truly independent. It also needs to be given the right authority, the right budget, and the right mandate in order to attract the right leadership.”

Rick Brennan, director of WHO’s emergency operations, found the panel’s report constructive. “Work has already begun on several of the recommendations, such as increasing staff and funds for emergency operations and integrating our health security and humanitarian work. I’m convinced that we will implement the rest of the recommendations, including the creation of a new WHO health emergency centre.”

Experts were unanimous on one point made in the report. With 20–30 cases occurring every week, Ebola in west Africa is not over and many eyes are now on WHO’s role in ending it.

EDITORIAL
A plan to protect the world—and save WHO
The Lancet July 11, 2015
The Lancet, Vol. 386, No. 9989, p103

“WHO must reestablish its pre-eminence as the guardian of global public health.” These words resonate throughout the final report of the Ebola Interim Assessment Panel, requested by WHO’s Executive Board, chaired by Dame Barbara Stocking, and published this week. The findings of the panel present a devastating critique of WHO and the chronic inaction of its member states, which together created the conditions for an Ebola virus disease outbreak of unprecedented ferocity and human tragedy. The Stocking Report, as it will come to be known, sets out in agonising detail how the entire global health system fatally let down the people of west Africa.

Stocking reserves her harshest criticism for WHO. The delays in announcing a Public Health Emergency of International Concern (it took 5 months from announcing an “unprecedented outbreak” in April, 2014, to declaring a public health emergency on August 8) was “unjustifiable”. The agency’s culture is unfit to manage an emergency response. Independent and courageous decision-making by the Director-General of WHO and her team “was absent in the early months of the Ebola crisis”. The agency was slow and reactive to events. WHO has lost its position as the authoritative body on health emergencies. It thought it could manage Ebola through polite behind-the-scenes international diplomacy. It failed to recognise that Ebola was a health emergency, not a diplomatic puzzle. And WHO’s communication strategy for Ebola simply “failed”. The agency failed to communicate proactively and it failed to establish itself as the authoritative voice on the Ebola outbreak. Member States of WHO are not spared. They have persistently failed to take the International Health Regulations (IHR, 2005) seriously—a position that is “irresponsible” and “untenable” for global health security. They should adopt the notion of “shared sovereignty”. They need to invest in WHO (the Panel proposes a modest 5% increase in assessed contributions in 2016).

The Panel’s recommendations are clear and forthright. Although WHO was severely criticised, Stocking argues that the agency should still take the lead for emergency health responses. But to do so, WHO must undergo “significant transformation”—not least, adequate funding and a change in culture. It must provide costed plans for establishing core public health capacities as set out in the IHR (2005). It should establish a new WHO Centre for Emergency Preparedness and Response, with an independent board that publishes a report on Global Health Security annually. WHO country and regional offices should be strengthened. The agency should take its role in accelerating the research and development of diagnostics, vaccines, and medicines more seriously. And WHO should do more to coordinate its activities with other parts of the humanitarian community. The IHR Review Committee should examine the value of an intermediate alert for a public health emergency, lowering the threshold at which the world can be warned of a new health risk. And sanctions against countries that violate the IHR should be considered.

The Panel makes clear that global health must be put at the centre of the global security agenda. But while its recommendations are cogent, there are three important omissions that deserve attention. First, the Panel does not address the vicious cycle within which WHO is caught. The reason why WHO is so poorly resourced is that it lacks the confidence of donors. As the agency continues to underperform because of chronic underinvestment, so that lack of confidence (and the resultant unwillingness to invest) only worsens. The Panel presents no way out of this endless circle of failure. Second, one of the most important responsibilities for governments is the preservation of public order and national security. In the context of Ebola (indeed, any health crisis), this means creating resilient health systems to protect populations from unexpected shocks, as explained by Mosoka Fallah and colleagues in a letter from Liberia’s Ministry of Health this week. Universal health coverage should have been emphasised as a crucial instrument in building global health security. Finally, the Panel rightly notes that, “While WHO has already accepted the need for transformation of its organisational culture and delivery, it will need to be held accountable to ensure that this transformation is achieved”. However, nowhere does the Panel recommend the accountability mechanism to monitor and review the implementation of its recommendations. Our fear is that the unique opportunity presented by the Stocking Report will be squandered. We have little confidence that the governing bodies of WHO will deliver on the expectations of Stocking and her team. The responsibility for action therefore falls to WHO’s Director-General. Dr Margaret Chan has 20 months to save her agency from further and possibly irreversible reputational damage.

ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study
Mara Jana Broadhurst, John Daniel Kelly, Ann Miller, Amanda Semper, Daniel Bailey, et al.

The Lancet, June 25, 2015; Vol. 386, No. 9996, p867–874    http://dx.doi.org/10.1016/S0140-6736(15)61042-X    
Background  At present, diagnosis of Ebola virus disease requires transport of venepuncture blood to field biocontainment laboratories for testing by real-time RT-PCR, resulting in delays that complicate patient care and infection control efforts. Therefore, an urgent need exists for a point-of-care rapid diagnostic test for this disease. In this Article, we report the results of a field validation of the Corgenix ReEBOV Antigen Rapid Test kit.
Methods   We performed the rapid diagnostic test on fingerstick blood samples from 106 individuals with suspected Ebola virus disease presenting at two clinical centres in Sierra Leone. Adults and children who were able to provide verbal consent or assent were included; we excluded patients with haemodynamic instability and those who were unable to cooperate with fingerstick or venous blood draw. Two independent readers scored each rapid diagnostic test, with any disagreements resolved by a third. We compared point-of-care rapid diagnostic test results with clinical real-time RT-PCR results (RealStar Filovirus Screen RT-PCR kit 1·0; altona Diagnostics GmbH, Hamburg, Germany) for venepuncture plasma samples tested in a Public Health England field reference laboratory (Port Loko, Sierra Leone). Separately, we performed the rapid diagnostic test (on whole blood) and real-time RT-PCR (on plasma) on 284 specimens in the reference laboratory, which were submitted to the laboratory for testing from many clinical sites in Sierra Leone, including our two clinical centres.
Findings   In point-of-care testing, all 28 patients who tested positive for Ebola virus disease by RT-PCR were also positive by fingerstick rapid diagnostic test (sensitivity 100% [95% CI 87·7–100]), and 71 of 77 patients who tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [95% CI 83·8–97·1]). In laboratory testing, all 45 specimens that tested positive by RT-PCR were also positive by the rapid diagnostic test (sensitivity 100% [95% CI 92·1–100]), and 214 of 232 specimens that tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [88·0–95·3]). The two independent readers agreed about 95·2% of point-of-care and 98·6% of reference laboratory rapid diagnostic test results. Cycle threshold values ranged from 15·9 to 26·3 (mean 22·6 [SD 2·6]) for the PCR-positive point-of-care cohort and from 17·5 to 26·3 (mean 21·5 [2·7]) for the reference laboratory cohort. Six of 16 banked plasma samples from rapid diagnostic test-positive and altona-negative patients were positive by an alternative real-time RT-PCR assay (the Trombley assay); three (17%) of 18 samples from individuals who were negative by both the rapid diagnostic test and altona test were also positive by Trombley.
Interpretation   The ReEBOV rapid diagnostic test had 100% sensitivity and 92% specificity in both point-of-care and reference laboratory testing in this population (maximum cycle threshold 26·3). With two independent readers, the test detected all patients who were positive for Ebola virus by altona real-time RT-PCR; however, this benchmark itself had imperfect sensitivity.
Malaria morbidity and mortality in Ebola-affected countries caused by decreased health-care capacity, and the potential effect of mitigation strategies: a modelling analysis
Patrick G T Walker, Michael T White, Jamie T Griffin, Alison Reynolds, Neil M Ferguson, Azra C Ghani
The Lancet Infectious Diseases, April 23, 2015; Vol. 15, No. 7, p825–832  http://dx.doi.org/10.1016/S1473-3099(15)70124-6    
Background  The ongoing Ebola epidemic in parts of west Africa largely overwhelmed health-care systems in 2014, making adequate care for malaria impossible and threatening the gains in malaria control achieved over the past decade. We quantified this additional indirect burden of Ebola virus disease.
Methods  We estimated the number of cases and deaths from malaria in Guinea, Liberia, and Sierra Leone from Demographic and Health Surveys data for malaria prevalence and coverage of malaria interventions before the Ebola outbreak. We then removed the effect of treatment and hospital care to estimate additional cases and deaths from malaria caused by reduced health-care capacity and potential disruption of delivery of insecticide-treated bednets. We modelled the potential effect of emergency mass drug administration in affected areas on malaria cases and health-care demand.
Findings  If malaria care ceased as a result of the Ebola epidemic, untreated cases of malaria would have increased by 45% (95% credible interval 43–49) in Guinea, 88% (83–93) in Sierra Leone, and 140% (135–147) in Liberia in 2014. This increase is equivalent to 3·5 million (95% credible interval 2·6 million to 4·9 million) additional untreated cases, with 10 900 (5700–21 400) additional malaria-attributable deaths. Mass drug administration and distribution of insecticide-treated bednets timed to coincide with the 2015 malaria transmission season could largely mitigate the effect of Ebola virus disease on malaria.
Interpretation  These findings suggest that untreated malaria cases as a result of reduced health-care capacity probably contributed substantially to the morbidity caused by the Ebola crisis. Mass drug administration can be an effective means to mitigate this burden and reduce the number of non-Ebola fever cases within health systems.
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Turning genetic information into working proteins

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series 2; 3.3

James E. Darnell Jr. (1930— )
Vincent Astor Professor Emeritus
2002 Albert Lasker Award for Special Achievement in Medical Science

Responsible for the various tasks required in turning genetic information into working proteins, ribonucleic acids are one of the most essential players in the life of a cell. First discovered in 1868, RNA today remains the subject of intense scientific scrutiny. Over the course of a career dedicated to understanding the intricate workings of gene transcription, Rockefeller University scientist James E. Darnell Jr. has revealed some of RNA’s most secretive and surprising mechanisms. For his half-century of illuminating research, Dr. Darnell received the 2002 Albert Lasker Award for Special Achievement in Medical Science.

In 1963, Dr. Darnell described a phenomenon he termed “RNA processing,” a step in the process of gene transcription, which had only recently been elucidated in bacterial systems. Working with mammalian cells — which differ from bacterial cells in that they contain a nucleus, where RNA is created — Dr. Darnell observed that very long strings of RNA disappear from the cell nucleus and that subsequently, shorter RNAs resembling the absent longer ones appear in the cytoplasm. Mammalian cells, he concluded, must distill their massive, immature nuclear RNA into shorter, mature forms that are individually coded for specific purposes by specific segments of the genome.

Dr. Darnell carried the principles of his finding — which he made in ribosomal RNA, part of the construction crew that builds cellular proteins — to other long nuclear RNA, including the longest one, which he named heterogeneous nuclear RNA (hnRNA). His hypothesis, that hnRNA is the precursor of the better known messenger RNA — which carries the genetic blueprint for protein building — soon bore fruit when he found a structural correlation between the two. Certain hnRNAs and nearly all messenger RNAs have a “tail” of adenine nucleotides at one end. Dr. Darnell followed this discovery with the observation that when an hnRNA string with an adenine tail disappears from the nucleus, a messenger RNA with the same tail then appears in the cytoplasm, suggesting a causal link between the two. When he found a second similarity — a cap at the end of the string opposite the adenine tail — he faced a conundrum. Scientific dogma had it that the order of nucleotides in any RNA mirrors that of DNA, whether the RNA is modeled from somewhere in the middle of the DNA or from one of the ends. The matching of a nuclear RNA to its cytoplasmic product by two end pieces glued together was surprising, but the concept was soon proven by colleagues at other institutions and called RNA splicing.

After a brief sojourn in Paris to work in François Jacob’s lab, Darnell worked at MIT, the Albert Einstein College of Medicine, and Rockefeller University on the relationship between mRNA and hnRNA. hnRNA was believed to be the precursor to mRNA, and despite making some key discoveries, Darnell admits that he could not free his imagination from the idea of colinearity and envision an hnRNA spliced to produce a smaller mRNA.

At this time, Darnell turned his attention to the question he had pondered since Paris: how were genes regulated in animal cells? This led to the discovery of the STAT and the Jak-STAT pathway of transcription control.

With the knowledge of RNA processing and splicing, Dr. Darnell next examined how cells begin the process of transcription and how they activate particular segments of DNA. Having moved to Rockefeller University in 1974, he found in the early 1980s that cells retain their specificity only in the context of their natural environment. Away from other liver cells, for example, a single liver cell stops producing liver-specific RNA, though it continues to make RNA for more generic cellular tasks. To pinpoint the signals responsible, which he believed must be coming from outside the cell, Dr. Darnell took a closer look at interferons (IFN), proteins that warn a cell when it’s time to raise its genetic defenses against harmful microbes.

Dr. Darnell’s laboratory studies how signals from the cell surface affect transcription of genes in the nucleus. Originally using interferon as a model cytokine, the Darnell group discovered that cell transcription was quickly changed by binding of cytokines to the cell surface. Introducing IFNβ into cell cultures, he watched as a particular type of mRNA accumulated in the cytoplasm, unaccompanied by any new protein synthesis. Analyzing the mRNA led him to the segment of DNA that had been activated, and the lack of new proteins told him that the cell contained its own, usually dormant, IFN-responsive transcription factor. By isolating a particular stretch of DNA from IFN-treated cells, he was able to call out of hiding the proteins that make up that factor, which, partly because they respond to signals very quickly, he called “STATs.” Dr. Darnell then traced the chemical relay that activates the STATs after IFN contact, called the Jak-Stat pathway.

The bound interferon led to the tyrosine phosphorylation of latent cytoplasmic proteins now called STATs (signal transducers and activators of transcription) that dimerize by reciprocal phosphotyrosine-SH2 interchange. They accumulate in the nucleus, bind DNA and drive transcription. This pathway has proved to be of wide importance, with seven STATs now known in mammals that take part in a wide variety of developmental and homeostatic events in all multicellular animals. Crystallographic analysis defined functional domains in the STATs, and current attention is focused on two areas: how the STATs complete their cycle of activation and inactivation, which requires regulated tyrosine dephosphorylation; and how persistent activation of STAT3 that occurs in a high proportion of many human cancers contributes to blocking apoptosis in cancer cells. Current efforts are devoted to inhibiting STAT3 with modified peptides that can enter cells.

 

Dr. Darnell received his M.D. in 1955 from the Washington University School of Medicine. His career has included poliovirus research with Harry Eagle at the National Institute of Allergy and Infectious Diseases, research with François Jacob at the Pasteur Institute in Paris and academic appointments at the Massachusetts Institute of Technology, the Albert Einstein College of Medicine and Columbia University. In 1974 Dr. Darnell joined Rockefeller as Vincent Astor Professor, and from 1990 to 1991 he was vice president for academic affairs.

A member of the National Academy of Sciences since 1973, he has received numerous awards, including the 2012 Albany Medical Center Prize in Medicine and Biomedical Research, the 2003 National Medal of Science, the 2002 Albert Lasker Award for Special Achievement in Medical Science, the 1997 Passano Award, the 1994 Paul Janssen Prize in Advanced Biotechnology and Medicine and the 1986 Gairdner Foundation International Award.

He is the coauthor with S.E. Luria of General Virology and the founding author with Harvey Lodish and David Baltimore of Molecular Cell Biology, now in its seventh edition. His book RNA, Life’s Indispensable Molecule was published in July 2011 by Cold Spring Harbor Laboratory Press. He is a member of the American Academy of Arts and Sciences and a foreign member of The Royal Society and The Royal Swedish Academy of Sciences.

 

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Viruses, Vaccines and Immunotherapy

Writer and Curator: Larry H. Bernstein, MD, FCAP

This discussion is exclusively concerned with viruses, the infectious particles of coated DNA or RNA, that have been a major cause of epidemics.  There will be a presentation of mechanisms of disease and disease resistance, and of diagnosis and treatment.  The preventive treatment of viral disease has a long history that began with the discovery by Edward Jenner (1772) that the milkmaids did not get “smallpox”, but were protected by a history of “cowpox”, which led to the vaccine (1796). Jenner’s work is widely regarded as the foundation of immunology.  Rabies may be the oldest infectious disease known to man.  Louis Pasteur created the first vaccine in 1885.
Today we appreciate the advances in vaccination as a matter of public health with – measles, mumps, rubella, and the astounding work on polio. The making of a vaccine for influenza has been an evolutionary struggle.

Edward Jenner

Edward Jenner

For many centuries, smallpox devastated mankind.  It was common knowledge that survivors of smallpox became immune to the disease. As early as 430 BC, survivors of smallpox were called upon to nurse the afflicted. Interestingly, reminiscent of Florence Nightingale, Dr. Sydenham (1624–1689) treated his patients by allowing no fire in the room, leaving the windows permanently open.  Prior to vaccination, innoculation or variolation was used by subcutaneous insertion by a lancet wet with fresh matter taken from a ripe pustule of some person who suffered from smallpox. This method was introduced by Circassian traders (to immunize the Sultan’s Harem as children)  in the Ottoman Turkish Empire in 1670 .

The earliest evidence of skin lesions resembling those of smallpox is found on faces of mummies from the time of the 18th and 20th Egyptian Dynasties (1570–1085 bc). The first stages of the decline of the Roman Empire (ad 108) coincided with a large-scale epidemic: the plague of Antonine, which accounted for the deaths of almost 7 million people. The Arab expansion, the Crusades, and the discovery of the West Indies all contributed to the spread of the disease. With the rapid pace of vaccine development in recent decades, the historic origins of immunization are often forgotten.

Unknown in the New World, smallpox was introduced by the Spanish and Portuguese conquistadors. The disease decimated the local population and was instrumental in the fall of the empires of the Aztecs and the Incas. Similarly, on the eastern coast of North America, the disease was introduced by the early settlers and led to a decline in the native population. The devastating effects of smallpox also gave rise to one of the first examples of biological warfare. During the French-Indian War (1754–1767), Sir Jeffrey Amherst, the commander of the British forces in North America, suggested the deliberate use of smallpox to diminish the American Indian population hostile to the British. Another factor contributing to smallpox in the Americas was the slave trade because many slaves came from regions in Africa where smallpox was endemic. Since the attack on the World Trade Center on September 11, 2001, the threat of biological warfare and bioterrorism has reemerged. Smallpox has been identified as a possible agent of bioterrorism.
(Google sources)

Hepatitis C/B

Virus-host interactomics: new insights and opportunities for antiviral
drug discovery
B de Chassey, L Meyniel-Schicklin, J Vonderscher1, P André and V Lotteau
de Chassey et al. Genome Medicine 2014, 6:115
http://genomemedicine.com/content/6/11/115

The current therapeutic arsenal against viral infections remains limited,
with often poor efficacy and incomplete coverage, and appears inadequate
to face the emergence of drug resistance. Our understanding of viral biology
and pathophysiology and our ability to develop a more effective antiviral
arsenal would greatly benefit from a more comprehensive picture of the
events that lead to viral replication and associated symptoms. Towards this
goal, the construction of virus-host interactomes is instrumental, mainly
relying on the assumption that a viral infection at the cellular level can be
viewed as a number of perturbations introduced into the host protein network
when viral proteins make new connections and disrupt existing ones. Here,
we review advances in interactomic approaches for viral infections, focusing
on high-throughput screening (HTS) technologies and on the generation of
high-quality datasets. We show how these are already beginning to offer
intriguing perspectives in terms of virus-host cell biology and the control of
cellular functions, and we conclude by offering a summary of the current
situation regarding the potential development of host-oriented antiviral
therapeutics.

Innate and adaptive immune responses in HCV infections
Markus H. Heim, Robert Thimme
Journal of Hepatology Update: Hepatitis C
J Hepatol 2014; 61: j S14–S25.

Hepatitis C virus has been identified a quarter of a decade ago as a
leading cause of chronic viral hepatitis that can lead to cirrhosis
and hepatocellular carcinoma. Only a minority of patients can
clear the virus spontaneously during acute infection. Elimination
of HCV during acute infection correlates with a rapid induction of
innate, especially interferon (IFN) induced genes, and a delayed
induction of adaptive immune responses. However, the majority
of patients is unable to clear the virus and develops viral persistence
in face of an ongoing innate and adaptive immune
response. The virus has developed several strategies to escape
these immune responses. For example, to escape innate
immunity, the HCV NS3/4A protease can efficiently cleave and
inactivate two important signalling molecules in the sensory
pathways that react to HCV pathogen-associated molecular patterns
(PAMPs) to induce IFNs, i.e., the mitochondrial anti-viral
signalling protein (MAVS) and the Toll-IL-1 receptor-domaincontaining
adaptor-inducing IFN-b (TRIF). Despite these escape
mechanisms, IFN-stimulated genes (ISGs) are induced in a large
proportion of patients with chronic infection. Of note, chronically
HCV infected patients with constitutive IFN-stimulated gene
(ISG) expression have a poor response to treatment with pegylated
IFN-a (PegIFN-a) and ribavirin. The mechanisms that protect
HCV from IFN-mediated innate immune reactions are not entirely
understood, but might involve blockade of ISG protein translation
at the ribosome, localization of viral replication to cell compartments
that are not accessible to anti-viral IFN-stimulated effector
systems, or direct antagonism of effector systems by viral
proteins. Escape from adaptive immune responses can be
achieved by emergence of viral escape mutations that avoid recognition
by antibodies and T cells. In addition, chronic infection is
characterized by the presence of functionally and phenotypically
altered NK and T cell responses that are unable to clear the virus
but most likely contribute to the ongoing liver disease. In this
review, we will summarize current knowledge about the role of
innate and adaptive immune responses in determining the outcome
of HCV infection.

Host-virus interactions in acute hepatitis C
Key Points
• Hepatitis C virus (HCV) has a very high replicative
capacity. Within days after infection, viral titres of
>106 IU/ml can be measured in the serum
• The innate immune system reacts to HCV infections
with the induction of interferon (IFN)-stimulated genes
in the liver. This initial type I and/or type III IFN driven
response controls viral replication to some extent, but
can not eliminate HCV completely
• 4-8 weeks after infection, HCV specific T cells are
recruited to the liver. HCV replication is inhibited by
noncytolytic (IFN-γ mediated) and cytolytic mechanisms.
In about 30% of patients, the immune reaction during
acute hepatitis C is strong enough to eliminate HCV
infection.
• In the acute phase of the infection, HCV is highly
vulnerable to therapy with recombinant IFN-α. Over
90% of patients can be cured with IFN-α monotherapy

Severity of Hepatitis C Virus (Genotype-3) Infection Positively
Correlates with Circulating MicroRNA-122 in Patients Sera
S Kumar, YK Chawla, S Ghosh, and A Chakraborti
Disease Markers 2014; Article ID 435476, 6 pages
http://dx.doi.org/10.1155/2014/435476

Introduction. Hepatitis C virus (genotype-3) causes acute and
chronic hepatitis infection predomination in India. The infectious
phase of the virus requires various host factors for its survival
and subsequent viral particle production. Small RNA molecules like
microRNA-122 (miR-122) are one such factormostly present in the
liver and play a supportive role in viral replication. Objective. In
this study, diagnostic potential of miR-122 is evaluated in the sera
of chronic hepatitis C patients. Methods. miRNAs were isolated
fromthe sera samples of patients as well as controls and miR-122
expression was quantified by real-time PCR. Results. A significant
augmentation was observed in the level of circulating miR-122
(median level, 0.66 versus 0.29, p = 0.001) in patients compared to
controls with ROC value of 0.929 ± 0.034 (p < 0.001). Interestingly,
miR-122 level also depicted a significant positive correlation
with serum ALT (p = 0.53), AST (p = 0.44), and viral load
(p = 0.52). Conclusion. The study thus unveiled the role of miR-122 as a
plausible diagnostic biomarker during HCV genotype-3 infection in India.

MicroRNA Expression Profiling in PBMCs: A Potential Diagnostic
Biomarker of Chronic Hepatitis C
Chiu-Chun Chang, Chun-Che Lin, Wan-Ling Hsieh, Hsin-Wu Lai, et al.
Disease Markers 2014; Article ID 367157, 9 pages
http://dx.doi.org/10.1155/2014/367157

The expression levels of miR-16, miR-193b, miR-199a, miR-222, and
miR-324 in PBMCs were significantly higher in CHC patients
compared with healthy controls and significantly different between
CHC patients withHCV genotype 1 (GT-1) and non-genotype-1
(non-GT-1).Multivariate logistic regression analysis also showed
that patients with high expression levels of the six target miRNAs
had an approximately 7.202-fold risk of CHC compared with those
with low expression levels of the target miRNAs.We concluded
that the expression levels of miR-16, miR-193b, miR-199a, miR-222,
and miR-324 target miRNAs in PBMCs of CHC may act as
significant risk biomarkers for the development of CHC.

Neutrophil-to-Lymphocyte Ratio as a Predictor of Response to
Peginterferon plus Ribavirin Therapy for Chronic Hepatitis C
Ming-Te Kuo, Tsung-Hui Hu, Sheng-Nan Lu, CH Hung, Jing-Houng Wang, et al.
Disease Markers 2014; Article ID 462958, 6 pages
http://dx.doi.org/10.1155/2014/462958

We aimed to determine whether neutrophil-to-lymphocyte ratio (NLR)
could be a predictor of antiviral response in chronic hepatitis C patients.
A total of 602 consecutive patients (genotype 1, n = 263; genotype 2,
n = 297; others/unknown, n = 42) receiving response-guided
therapy with peginterferon plus ribavirin were recruited. NLR was
related to clinical and virological features and to treatment outcome.
Rapid virological response (RVR) and sustained virological response
(SVR) were achieved in 436 (73%) and 458 (76%) of the patients,
respectively. Higher NLR ( ≥1.42) was found to be associated with
higher prevalence of DM (p = 0.039) and higher hepatitis C viral
load (p = 0.002) and white cell count (p < 0.001). NLR was
significantly lower in patients with RVR and SVR compared
to those without (𝑃 = 0.032 and 0.034, resp.). However, NLR
was not an independent factor by multivariate analysis. In the
subgroup analysis, higher NLR (≥1.42) (odds ratio, 0.494,
p = 0.038) was an independent poor predictor of SVR in
genotype 2 patients but was not in genotype 1 patients. In
conclusion, NLR is a simple and easily accessible marker to
predict response to peginterferon plus ribavirin therapy
for chronic hepatitis C genotype 2.

Therapy of Hepatitis C — Back to the Future
T. Jake Liang and Marc G. Ghany
N Engl J Med  http://nejm.org

The results of several phase 3 studies of interferon-free combination
regimens of DAAs reported in the Journal now2-4 and recently5-9
unequivocally show the superiority of two regimens over the standard-
of-care treatment (a combination of peginterferon, ribavirin, and
a protease inhibitor) for HCV genotype 1 infection. A previous editorial
in the Journal highlighted the significantly improved response rates
(rates of sustained virologic response of 93% to 99%) with a
coformulated regimen of sofosbuvir (a nucleotide NS5B inhibitor)
and ledipasvir (an NS5A inhibitor) among patients with HCV genotype
1 infection, as compared with the rates with the previously approved
interferon-based single-DAA combination therapy.

Sofosbuvir and Ribavirin in HCV Genotypes 2 and 3
S Zeuzem, GM Dusheiko, R Salupere, et al.
N Eng J Med May 2014  http://dx.doi.org:/10.1056/NEJMoa1316145

Background
In clinical trials, treatment with a combination of the nucleotide
polymerase inhibitor sofosbuvir and the antiviral drug ribavirin was
associated with high response rates among patients with hepatitis C
virus (HCV) genotype 2 infection, with lower response rates among
patients with HCV genotype 3 infection.
Methods
We conducted a study involving patients with HCV genotype 2 or 3
infection, some of whom had undergone previous treatment with
an interferon-based regimen. We randomly assigned 91 patients with
HCV genotype 2 infection and 328 with HCV genotype 3 infection, in
a 4:1 ratio, to receive sofosbuvir–ribavirin or placebo for 12 weeks.
On the basis of emerging data from phase 3 trials indicating that
patients with HCV genotype 3 infection had higher response rates
when they were treated for 16 weeks, as compared with 12 weeks,
the study was unblinded, treatment for all patients with genotype 3
infection was extended to 24 weeks, the placebo group was terminated,
and the goals of the study were redefined to be descriptive and not
include hypothesis testing. The primary end point was a sustained
virologic response at 12 weeks after the end of therapy.
Results
Of the 419 patients who were enrolled and treated, 21% had cirrhosis
and 58% had received previous interferon-based treatment. The criterion
for a sustained virologic response was met in 68 of 73 patients (93%; 95%
confidence interval [CI], 85 to 98) with HCV genotype 2 infection who
were treated for 12 weeks and in 213 of 250 patients (85%; 95% CI, 80 to 89)
with HCV genotype 3 infection who were treated for 24 weeks. Among
patients with HCV genotype 3 infection, response rates were 91%
and 68% among those without and those with cirrhosis, respectively.
The most common adverse events were headache, fatigue, and pruritus.
Conclusions
Therapy with sofosbuvir–ribavirin for 12 weeks in patients with HCV
genotype 2 infection and for 24 weeks in patients with HCV genotype 3
infection resulted in high rates of sustained virologic response. (Funded
by Gilead Sciences; VALENCE ClinicalTrials.gov number, NCT01682720.)

New developments in the management of hepatitis C virus infection:
focus on boceprevir
Marina Berenguer, F Xavier López-Labrador
Biologics: Targets and Therapy 2012:6 249–256
http://dx.doi.org/10.2147/BTT.S24413

Chronic hepatitis C virus infection is an important public health problem,
and the standard treatment (combination of pegylated interferon-α and
ribavirin) has an effectiveness rate of only 40%–50%. Novel virus-specific
drugs have recently been designed, and multiple compounds are under
development. The approval for the clinical use of direct-acting antivirals
in 2011 (boceprevir [BOC] and telaprevir, viral NS3 protease inhibitors)
has increased recovery rates by up to 70%. Therefore, a highly effective
treatment has been envisioned for the first time. This paper focuses on
BOC and the implementation of new BOC-based treatment regimes.

ABT-450/r–Ombitasvir and Dasabuvir with or without Ribavirin for HCV
P Ferenci, D Bernstein, J Lalezari, D Cohen, Y Luo, C Cooper, et al.
N Engl J Med May 4, 2014. http://dx.doi.org:/10.1056/NEJMoa1402338

Background
The interferon-free regimen of ABT-450 with ritonavir (ABT-450/r),
ombitasvir, and dasabuvir with or without ribavirin has shown efficacy
in inducing a sustained virologic response in a phase 2 study involving
patients with hepatitis C virus (HCV) genotype 1 infection. We conducted
two phase 3 trials to examine the efficacy and safety of this regimen in
previously untreated patients with HCV genotype 1 infection and no cirrhosis.
Methods
We randomly assigned 419 patients with HCV genotype 1b infection
(PEARL-III study) and 305 patients with genotype 1a infection (PEARL-IV
study) to 12 weeks of ABT-450/r–ombitasvir (at a once-daily dose of
150 mg of ABT-450, 100 mg of ritonavir, and 25 mg of ombitasvir), dasabuvir
(250 mg twice daily), and ribavirin administered according to body weight
or to matching placebo for ribavirin. The primary efficacy end point was a
sustained virologic response (an HCV RNA level of <25 IU per milliliter)
12 weeks after the end of treatment.
Results
The study regimen resulted in high rates of sustained virologic response
among patients with HCV genotype 1b infection (99.5% with ribavirin
and 99.0% without ribavirin) and among those with genotype 1a infection
(97.0% and 90.2%, respectively). Of patients with genotype 1b infection,
1 had virologic failure, and 2 did not have data available at post-treatment
week 12. Among patients with genotype 1a infection, the rate of virologic
failure was higher in the ribavirin-free group than in the ribavirin
group (7.8% vs. 2.0%). In both studies, decreases in the hemoglobin
level were significantly more common in patients receiving ribavirin.
Two patients (0.3%) discontinued the study drugs owing to adverse events.
The most common adverse events were fatigue, headache, and nausea.
Conclusions
Twelve weeks of treatment with ABT-450/r–ombitasvir and dasabuvir
without ribavirin was associated with high rates of sustained virologic
response among previously untreated patients with HCV genotype 1
infection. Rates of virologic failure were higher without ribavirin than
with ribavirin among patients with genotype 1a infection but not among
those with genotype 1b infection. (Funded by AbbVie; PEARL-III and
PEARL-IV ClinicalTrials.gov numbers, NCT01767116 and NCT01833533.)

Individualized treatment of chronic hepatitis C with pegylated interferon
and ribavirin
Roberto J Carvalho-Filho and Olav Dalgard
Pharmacogenomics and Personalized Medicine 2010:3 1–13

Chronic infection with hepatitis C virus (HCV) is a major public health
problem, with perhaps 180 million people infected worldwide. A significant
proportion of these will eventually develop clinical complications, such as
cirrhosis, liver decompensation and hepatocellular carcinoma. Sustained
virological response (SVR) to antiviral therapy is associated with
improvement in liver histology and survival free of liver-related complications.
Great effort has been made to improve SVR rate by adapting the duration
of therapy according to HCV genotype and to on-treatment response. Rapid
virological response (RVR, undetectable HCV RNA at week 4) usually has
a high positive predictive value for achieving SVR and early virological
response (EVR, > 2 log reduction or undetectable HCV RNA at week 12)
exhibits a high negative predictive value for non-response. Individualized
approach can improve cost-effectiveness of HCV antiviral therapy by
reducing side effects and the costs of therapy associated with unnecessary
exposure to treatment and through extending therapy for those with
unfavorable features. This article summarizes recent data on strategies
of individualized treatment in naïve patients with mono-infection by
the different HCV genotypes. The management of common side effects,
the impact of HCV infection on health-related quality of life and the
potential applications of host genomics in HCV therapy are briefly discussed.

Simeprevir for the treatment of hepatitis C virus infection
L Izquierdo, F Helle, C François, S Castelain, G Duverlie, E Brochot
Pharmacogenomics and Personalized Medicine 2014:7 241–249
http://dx.doi.org/10.2147/PGPM.S52715

Simeprevir (TMC435, Olysio™), a second-generation hepatitis C virus
(HCV) protease inhibitor, has been recently approved for the treatment
of genotype 1 chronic hepatitis C in combination with pegylated interferon
and ribavirin. This molecule has very different characteristics from first-
generation protease inhibitors. Results from trials show that simeprevir
is highly effective and safe, with few adverse events. We discuss the
specific features of this new treatment option for HCV infection, in terms
of in vitro data, pharmacological data, and clinical trials. We also discuss
the impact of Q80K polymorphism at baseline. Studies evaluating interferon-
free regimens with simeprevir are ongoing. Future combinations of two or
more direct-acting antiviral agents, targeting different viral enzymes and
with synergistic antiviral effects, will be approved, allowing treatment of
pan-genotypic HCV with optimized sustained virologic responses. Simeprevir
will undoubtedly be part of future treatment strategies.

Boceprevir and personalized medicine in hepatitis C virus infection
F Habersetzer, C Leboeuf, M Doffoël, TF Baumert
Pharmacogenomics and Personalized Medicine 2012:5 125–137
http://dx.doi.org/10.2147/PGPM.S24259

Boceprevir was the first agent, along with telaprevir, of a novel class of
direct-acting antivirals that entered clinical practice for the treatment
of chronic hepatitis C. Boceprevir is an antiprotease that directly blocks
hepatitis C virus (HCV) replication. Two studies in patients with HCV
genotype 1 infection have shown that addition of boceprevir to the
standard of care, ie, pegylated interferon-alfa (PEG-IFN-α) and ribavirin,
markedly increased the rate of sustained virological response. A sustained
virological response was obtained in about 70% of patients who had never
been treated, as well as in 69%–75% and 40% of previous relapsers and
nonresponders to PEG-IFN-α-ribavirin, respectively. Side effects were
observed in almost all treated patients. Anemia, the most frequent adverse
event related to administration of boceprevir, occurred in about 50% of
patients. The decision to add boceprevir to the standard of care is made
on an individual basis, and takes into account the prognosis of the liver
disease, the efficacy of therapy, as it could be at best predicted, and the
side effects that may arise, taking into account the comorbidities of the
patient. Ultimately, the treatment must be accepted by the patient, who
should fully understand the benefits and risks. Boceprevir trials were
designed with the concept of individualized and response-guided therapy
which establishes treatment decisions on how rapidly patients respond
to treatment. Individualized therapy for chronic hepatitis C is based on
patient and viral characteristics to make the best choice about whether a
person will benefit from therapy and to evaluate on-treatment predictors
of response to shorten therapy in patients with a rapid response as well as
in patients who did not respond sufficiently to expect HCV eradication.
This review focuses on the main results obtained so far, their impact on
the treatment of patients with chronic hepatitis C, and potential
therapeutic perspectives.

Ribavirin at the Era of Novel Direct Antiviral Agents for the Treatment
of Hepatitis C Virus Infection: Relevance of Pharmacological Monitoring
P Pradat, V Virlogeux, Marie-Claude Gagnieu, F Zoulim, and F Bailly
Adv Hepatol 2014, Article ID 493087, 13 pages
http://dx.doi.org/10.1155/2014/493087

Ribavirin is often used for the treatment of hepatitis C virus (HCV)
infection. Although its mechanisms of action remain to be clearly
elucidated, ribavirin plays a beneficial role for achieving virological
response and decreasing the rate of virological relapse after
treatment cessation. However, ribavirin may induce side effects
leading to early treatment discontinuation. Among them,
hemolytic anemia is the most frequent and results from
intraerythrocyte accumulation. Pharmacological studies have
shown that early ribavirin exposure assessed by the area under
the curve (AUC) at day 0 and ribavirin trough concentration
during the first three months of therapy were correlated with
sustained virological response (SVR). These studies highlighted
the relevance of ribavirin pharmacologic monitoring and early
dose adaptation during therapy. Although the role of ribavirin
within new direct acting antiviral (DAA) combinations will
probably decrease in the future, its potential benefit in difficult-
to-treat patients such as patients with severe hepatopathy or
patients who failed triple therapy including patients with
multiresistance will need to be further investigated.

Clinical utility of pharmacogenomics in the management
of hepatitis C
J Trinks, ML Hulaniuk, MA Redal, D Flichman
Pharmacogenomics and Personalized Medicine 2014:7 339–347
http://dx.doi.org/10.2147/PGPM.S52624

Hepatitis C virus (HCV) was identified for the first time more than
20 years ago. Since then, several studies have highlighted the
complicated aspects of this viral infection in relation to its worldwide
prevalence, its clinical presentation, and its therapeutic response.
Recently, two landmark scientific breakthroughs have moved us
closer to the successful eradication of chronic HCV infection. First,
response rates in treatment-naïve patients and in prior non-responders
to pegylated-interferon-α and ribavirin therapy are increasing as a direct
consequence of the development of direct-acting antiviral drugs. Secondly,
the discovery of single-nucleotide polymorphisms near the interleukin
28B gene significantly related to spontaneous and treatment-induced
HCV clearance represents a milestone in the HCV therapeutic landscape.
The implementation of this pharmacogenomics finding as a routine test
for HCV-infected patients has enhanced our understanding of viral
pathogenesis, has encouraged the design of ground-breaking antiviral
treatment regimens, and has become useful for pretreatment decision
making. Nowadays, interleukin 28B genotyping is considered to be a
key diagnostic tool for the management of HCV-infected patients and
will maintain its significance for new combination treatment schemes
using direct-acting antiviral agents and even in interferon-free regimens.
Such pharmacogenomics insights represent a challenge to clinicians,
researchers, and health administrators to transform this information
into knowledge with the aim of elaborating safer and more effective
therapeutic strategies specifically designed for each patient. In conclusion,
the individualization of treatment regimens for patients with hepatitis C,
that may lead to a universal cure in future years, is becoming a reality
due to recent developments in biomarker and genomic medicine.
In light of these advances, we review the scientific evidence and clinical
implications of recent findings related to host genetic factors in the
management of HCV infection.

Hepatitis B virus and Homo sapiens proteome-wide analysis: A
profusion of viral peptide overlaps in neuron-specific human proteins
Rosalia Ricco and Darja Kanduc
Biologics: Targets & Therapy 2010:4 75–81

The primary amino acid sequence of the hepatitis B virus (HBV)
proteome was searched for identity spots in the human proteome
by using the Protein Information Resource database. We find that
the HBV polyprotein shares sixty-five heptapeptides, one octapeptide,
and one nonapeptide with the human proteins. The viral matches
are disseminated among fundamental human proteins such as
adhesion molecules, leukocyte differentiation antigens, enzymes,
proteins associated with spermatogenesis, and transcription factors.
As a datum of special interest, a number of peptide motifs are shared
between the virus- and brain-specific antigens involved in neuronal
protection. This study may help to evaluate the potential cross
reactions and side effects of HBV antigen-based vaccines.

Dual effects of interleukin-18: inhibiting hepatitis B virus replication
in HepG2.2.15 cells and promoting hepatoma cells metastasis
Y Zhang, Y Li, Y Ma, S Liu, Y She, P Zhao, M Jing, et al.
Am J Physiol Gastrointest Liver Physiol 301: G565–G573, 2011
http://dx.doi.org:/10.1152/ajpglp.00058.2011

Interleukin-18 (IL-18) has been reported to inhibit hepatitis B
virus (HBV) replication in the liver of HBV transgenic mice;
however, the molecular mechanism of its antiviral effect has not
been fully understood. In the present study, it was shown that
IL-18 and its receptors (IL-18R) were constitutively expressed
in hepatoma cell lines HepG2 and HepG2.2.15 as well as
normal liver cell line HL-7702. We demonstrated that IL-18
directly inhibited HBV replication in HepG2.2.15 cells via
downregulating the activities of HBV core and X gene promoters.
The suppressed HBV replication by IL-18 could be rescued by
the administration of BAY11-7082, an inhibitor of transcription
factor NF-B. On the other hand, it was of interest that IL-18
promoted HepG2 cell metastasis and migration dose dependently
in both wound-healing assays and Transwell assays. The underlying
mechanism could be partially attributable to the increased activities
of extracellular matrix metalloproteinase (MMP)-9, MMP-3, and
MMP-2 by IL-18, which upregulated the mRNA levels of MMP-3
and MMP-9 in a NF-B-dependent manner. Furthermore, it was
confirmed that expression of IL-18/IL-18R and most MMPs were
remarkably upregulated in hepatocellular carcinoma (HCC) liver
cancer tissue specimens, suggesting that IL-18/IL-18R-triggered
signaling pathway was closely related to HCC metastasis
in vivo. Therefore, we revealed the dual effects of IL-18 in
human hepatocytes: it not only inhibited HBV replication but
also promoted hepatoma cells metastasis and migration. NF-B
played a critical role in both effects. Our work contributed to a
deeper understanding of the biological function of IL-18 in
human hepatocytes.

HIV

The export receptor Crm1 forms a dimer to promote nuclear
export of HIV RNA
David S Booth, Yifan Cheng, Alan D Frankel
eLife 2014;3:e04121 http://dx.doi.org:/10.7554/eLife.04121

The HIV Rev protein routes viral RNAs containing the Rev Response
Element (RRE) through the Crm1 nuclear export pathway to the
cytoplasm where viral proteins are expressed and genomic RNA is
delivered to assembling virions. The RRE assembles a Rev oligomer
that displays nuclear export sequences (NESs) for recognition by
the Crm1-RanGTP nuclear receptor complex. Here we provide the
first view of an assembled HIV-host nuclear export complex using
single particle electron microscopy. Unexpectedly, Crm1 forms a
dimer with an extensive interface that enhances association with
Rev-RRE and poises NES binding sites to interact with a Rev oligomer.
The interface between Crm1 monomers explains differences between
Crm1 orthologs that alter nuclear export and determine cellular tropism
for viral replication. The arrangement of the export complex identifies
a novel binding surface to possibly target an HIV inhibitor and may point
to a broader role for Crm1 dimerization in regulating host gene expression.

RNA-directed remodeling of the HIV-1 Rev protein orchestrates assembly
of the Rev-Rev response element complex
B Jayaraman, DC Crosby, C Homer, I Ribeiro, D Mavor, AD Frankel
eLife 2014; http://dx.doi.org/10.7554/eLife.04120

The HIV-1 protein Rev controls a critical step in viral replication by
mediating the nuclear export of unspliced and singly-spliced viral
mRNAs. Multiple Rev subunits assemble on the Rev Response Element
(RRE), a structured region present in these RNAs, and direct their
export through the Crm1 pathway. Rev-RRE assembly occurs via
several Rev oligomerization and RNA-binding steps, but how these
steps are coordinated to form an export-competent complex is unclear.
Here, we report the first crystal structure of a Rev dimer-RRE complex,
revealing a dramatic rearrangement of the Rev-dimer upon RRE binding
through re-packing of its hydrophobic protein-protein interface. Rev-RNA
recognition relies on sequence-specific contacts at the well-characterized
IIB site and local RNA architecture at the second site. The structure
supports a model in which the RRE utilizes the inherent plasticity of
Rev subunit interfaces to guide the formation of a functional complex.

HIV-1 envelope glycoprotein structure
Alan Merk and Sriram Subramaniam
Curr Opin Struct Biol. 2013 April ; 23(2): 268–276.
http://dx.doi.org:/10.1016/j.sbi.2013.03.007

The trimeric envelope glycoprotein of HIV-1, composed of gp120 and
gp41 subunits, remains a major target for vaccine development. The
structures of the core regions of monomeric gp120 and gp41 have
been determined previously by X-ray crystallography. New insights
into the structure of trimeric HIV-1 envelope glycoproteins are now
coming from cryo-electron tomographic studies of the gp120/gp41
trimer as displayed on intact viruses and from cryo-electron microscopic
studies of purified, soluble versions of the ectodomain of the trimer.
Here, we review recent developments in these fields as they relate to
our understanding of the structure and function of HIV-1 envelope
glycoproteins.

The QSAR and docking calculations of fullerene derivatives as HIV-1
protease inhibitors
Noha A. Saleh
Spectrochim Acta Part A: Molec and Biomol Spectrosc 2015; 136: 1523–1529
http://dx.doi.org/10.1016/j.saa.2014.10.045

The inhibition of HIV-1 protease is considered as one of the most
important targets for drug design and the deactivation of HIV-1.
In the present work, the fullerene surface (C60) is modified by
adding oxygen atoms as well as hydroxymethylcarbonyl (HMC)
groups to form 6 investigated fullerene derivative compounds.
These compounds have one, two, three, four or five O atoms +
HMC groups at different positions on phenyl ring. The effect of
the repeating of these groups on the ability of suggested compounds
to inhibit the HIV protease is studied by calculating both Quantitative
Structure Activity Relationship (QSAR) properties and docking simulation.
Based on the QSAR descriptors, the solubility and the hydrophilicity
of studied fullerene derivatives increased with increasing the number
of oxygen atoms + HMC groups in the compound. While docking
calculations indicate that, the compound with two oxygen atoms +
HMC groups could interact and binds with HIV-1 protease active site.
This is could be attributed to the active site residues of HIV-1 protease
are hydrophobic except the two aspartic acids. So that, the increase in the
hydrophilicity and polarity of the compound is preventing and/or
decreasing the hydrophobic interaction between the compound and
HIV-1 protease active site.

In silico identification of Novel HIVProtease inhibitors (PIs) using
ZINC drug Database
KK Srivastava, S Srivastava, T Alam, Rituraj
Int J Pharma Sci and Res (IJPSR) Dec 2014; 5(12):947-952.

The Human immunodeficiency virus type-1 protease is one of the
most important target of highly active anti-retrovirus therapy
(HAART) for the treatment of all acquired immune deficiency
syndrome (AIDS). Protease inhibitor Darunavir is most recent
included as a PI in the list of HARRT, more effective against mutant
type and wild type of Protease with increased no. of H-bonding then
precursors approved by FDA, So herein we taken Darunavir as a base
structure for virtually identification of more/similar efficient drug
like leads then Darunavir using PDB structure (3BGR) of Protease
from PDB database ‘RCSB’ versus chemical compounds database
‘ZINC’ using Schrodinger and Discovery Studio software. Using
molecular constraint search with similarity coefficient ‘Tanimoto’,
1,65,000 ligands were extracted and docking analysis resulted in
some efficient in docking and in other computational medicinal
parameters, we are reporting such leads, and, they may further
undergo through high end extensive virtual investigation and beyond.

DARUNAVIR DOCKED IN PDB 3BGR

DARUNAVIR DOCKED IN PDB 3BGR

In this work, we have tried to recognized some more/similar
potent drug like leads instead ‘Darunavir’ may be more effective,
we used five different RT crystallographic structures for better
identification/verification for our results, ZINC78487241,
ZINC09060710, ZINC72320180, ZINC78487244 & ZINC78487242
are showing very fine computed properties therefore, this study
verifies the importance of small drug like molecules libraries as
like ‘ZINC.docking.org’and their use certainly help scientific groups
to enhance their capabilities in drug discovery with reducing time,
including drug discovery process prior synthesis. Meanwhile all
herein identified molecules may further investigate instead “in silico”.

Development and Customization of a Color-Coded Microbeads-Based
Assay for Drug Resistance in HIV-1 Reverse Transcriptase
L Gu, Ai Kawana-Tachikawa, T Shiino, H Nakamura, M Koga, et al.
PLoS ONE 9(10): e109823. http://dx.doi.org:/10.1371/journal.pone.0109823

Other Virus Diseases

A global phylogenetic analysis in order to determine the host species
and geography dependent features present in the evolution of avian
H9N2 influenza hemagglutinin
Andrew R. Dalby and Munir Iqbal
PeerJ 2014. http://dx.doi.org:/10.7717/peerj.655

A complete phylogenetic analysis of all of the H9N2 hemagglutinin
sequences that were collected between 1966 and 2012 was carried out
in order to build a picture of the geographical and host specific evolution
of the hemagglutinin protein. To improve the quality and applicability
of the output data the sequences were divided into subsets based upon
location and host species. The phylogenetic analysis of hemagglutinin
reveals that the protein has distinct lineages between China and the
Middle East, and that wild birds in both regions retain a distinct form
of the H9 molecule, from the same lineage as the ancestral hemagglutinin.
The results add further evidence to the hypothesis that the current
predominant H9N2 hemagglutinin lineage might have originated in
Southern China. The study also shows that there are sampling problems
that affect the reliability of this and any similar analysis. This raises
questions about the surveillance of H9N2 and the need for wider
sampling of the virus in the environment. The results of this analysis
are also consistent with a model where hemagglutinin has predominantly
evolved by neutral drift punctuated by occasional selection events. These
selective events have produced the current pattern of distinct lineages in
the Middle East, Korea and China.

Describing the hexapeptide identity platform between the influenza A
H5N1 and Homo sapiens proteomes
Darja Kanduc
Biologics: Targets & Therapy 2010:4 245–261
http://dx.doi.org:/10.2147/BTT.S12097

We searched the primary sequence of influenza A H5N1 polyprotein
for hexamer amino acid sequences shared with human proteins using
the Protein International Resource database and the exact peptide
matching analysis program. We find that the viral polyprotein shares
numerous hexapeptides with the human proteome. The human
proteins involved in the viral overlap are represented by antigens
associated with basic cell functions such as proliferation, development,
and differentiation. Of special importance, many human proteins
that share peptide sequences with influenza A polyprotein are
antigens such as reelin, neurexin I-α, myosin-IXa, Bardet–Biedl
syndrome 10 protein, Williams syndrome transcription factor,
disrupted in schizophrenia 1 protein, amyotrophic lateral sclerosis 2
chromosomal region candidate gene 17 protein, fragile X mental
retardation 2 protein, and jouberin. That is, the viral-vs-human
overlap involves human proteins that, when altered, have been
reported to be potentially associated with multiple neurological
disorders that can include autism, epilepsy, obesity, dystonia,
ataxia–telangiectasia, amyotrophic lateral sclerosis, sensorineural
deafness, sudden infant death syndrome, Charcot-Marie-Tooth
disease, and myelination. The present data are discussed as a
possible molecular basis for understanding influenza A viral
escape from immunosurveillance and for defining anti-influenza
immune-therapeutic approaches devoid of collateral adverse events.

Middle East respiratory syndrome coronavirus neutralising serum
antibodies in dromedary camels: a comparative serological study

CBEM Reusken, BL Haagmans, MA Müller, C Gutierrez,
Gert-Jan Godeke, et al.
Lancet Infectious Diseases Oct 2013; 13(10): 859 – 866
http://dx.doi.org:/10.1016/S1473-3099(13)70164-6

A new betacoronavirus—Middle East respiratory syndrome coronavirus
(MERS-CoV)—has been identified in patients with severe acute
respiratory infection. Although related viruses infect bats, molecular
clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in
contact with dromedary camels or goats. We investigated possible
animal reservoirs of MERS-CoV by assessing specific serum antibodies
in livestock.
We took sera from animals in the Middle East (Oman) and from
elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40),
goats (n=40), dromedary camels (n=155), and various other camelid
species (n=34) were tested for specific serum IgG by protein microarray
using the receptor-binding S1 subunits of spike proteins of MERS-CoV,
severe acute respiratory syndrome coronavirus, and human coronavirus
OC43. Results were confirmed by virus neutralization tests for MERS-CoV
and bovine coronavirus.  50 of 50 (100%) sera from Omani camels and 15
of 105 (14%) from Spanish camels had protein-specific antibodies against
MERS-CoV spike. Sera from European sheep, goats, cattle, and other
camelids had no such antibodies. MERS-CoV neutralising antibody titers
varied between 1/320 and 1/2560 for the Omani camel sera and between
1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-
neutralization by bovine coronavirus antibodies. MERS-CoV or a related
virus has infected camel populations. Both titres and seroprevalences in
sera from different locations in Oman suggest widespread infection.

Cosic’s Resonance Recognition Model for Protein Sequences and
Photon Emission Differentiates Lethal and Non-Lethal Ebola
Strains: Implications for Treatment
Nirosha J. Murugan, Lukasz M. Karbowski, Michael A. Persinger
Open Journal of Biophysics, 2015, 5, 35-43
http://dx.doi.org/10.4236/ojbiophy.2015.51003

The Cosic Resonance Recognition Model (RRM) for amino acid
sequences was applied to the classes of proteins displayed by four
strains (Sudan, Zaire, Reston, Ivory Coast) of Ebola virus that
produced either high or minimal numbers of human fatalities.
The results clearly differentiated highly lethal and non-lethal strains.
Solutions for the two lethal strains exhibited near ultraviolet (~230 nm)
photon values while the two asymptomatic forms displayed near i
nfrared (~1000 nm) values. Cross-correlations of spectral densities of
the RRM values of the different classes of proteins associated with the
genome of the viruses supported this dichotomy. The strongest coefficient
occurred only between Sudan-Zaire strains but not for any of the other
pairs of strains for sGP, the small glycoprotein that intercalated with
the plasma cell membrane to promote insertion of viral contents into
cellular space. A surprising, statistically significant cross-spectral
correlation occurred between the “spike” glycoprotein component (GP1)
of the virus that associated the anchoring of the virus to the mammalian
cell plasma membrane and the Schumann resonance of the earth whose
intensities were determined by the incidence of equatorial thunderstorms.
Previous applications of the RRM to shifting photon wavelengths emitted
by melanoma cells adapting to reduced ambient temperature have validated
Cosic’s model and have demonstrated very narrow wave-length (about 10 nm)
specificity. One possible ancillary and non-invasive treatment of people
within which the fatal Ebola strains are residing would be whole body
application of narrow band near-infrared light pulsed as specific
physiologically-patterned sequences with sufficient radiant flux density
to perfuse the entire body volume.

Nov 23rd, 2014
Replication of Ebola Virus

Ebola Virus do not replicate through any kind of cell division; rather,
they use a combination of host and virally encoded enzymes, alongside
host cell structures, to produce multiple copies of viruses. These then self-assemble into viral macromolecular structures in the host cell. The
virus completes a set of steps when infecting each individual cell.

Replication of Ebola Virus

Following are the steps during the replication of Ebola Virus:

Attachment

First of all, there is attachment of virus to host receptors through
GP glycoprotein which is endocytosed into vesicles in the host cell.
Host DC-SIGN and DC-SIGNR play a role in virion attachment.

Viral Entry (Penetration)

The virion enters early endosomes by Macropinocytosis or
clathrin-mediated endocytosis.

Macropinocytosis

In this process, ruffled segments of the host’s plasma membrane
protrude outward from the cell and form invaginations where
the virus utilizes glycoproteins in order to attach to the surface
of the plasma membrane. Macropinocytosis is a process in which
the Eukaryotic host cells form macropinosomes, segments of plasma
membranes that extend out from the cell approximately 0.2-10 µm, in
order to incorporate the virus into the cell. The formation of macro-
pinosomes occurs spontaneously, as a result of the activation of various
growth factors, or simultaneously with the intake of cellular molecules
or extracellular fluid.

Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is the other means by which Ebolavirus
enters the host cell. This process is very similar to macropinocytosis
in that the plasma membrane forms invaginations that engulf the cell.
However, clathrin-mediated endocytosis is different in that proteins on
the surface of the host’s surface, and in particular clathrin, facilitate
the attachment of the virus to the host’s cell surface. Glycoproteins
are still used to attach the virus to the cell surface, and the NP-C1
cholesterol transporter still facilitates the fusion of the virus with
endosomes and lysosomes and still allows the virus to escape into
the cytoplasm. Without the NPC1 cholesterol transporter, Ebolavirus
cannot leave the vesicle in order to replicate and cause infection in other cells.

To penetrate the cell, the viral membrane fuses with vesicle membrane,
and the nucleocapsid is released into the cytoplasm.

In some culture cells, GP glycoprotein can be processed by host
Cathepsin L andCathepsin B into 19kDa GP1. But this processing is
not happening in all cells or for all ebolavirus. 19kDA GP1 interacts
with host NPC1, which is highly expressed in dendritic cells.

Fusion of virus membrane with the vesicle membrane is triggered
by either low pH orNPC1 binding.

Sequential Transcription

During transcription, the RNA genome is transcribed into seven
monocistronic mRNAs whose length is determined by highly
conserved start and stop signals.

The transcription process begins with the binding of the polymerase
complex to a single binding site located within the leader region of
the genome. The complex then slides along the RNA template and
sequentially transcribes the individual genes in their 3’ to 5’ order.
Encapsidated, negative-sense genomic ssRNA is used as a template
for the synthesis (3′-5′) of polyadenylated, monocistronic mRNAs
and, using the host cell’s ribosomes, tRNA molecules, etc.,
the mRNA is translated into individual viral proteins.

Replication

As viral protein levels rise, a switch occurs from translation to replication.
Using the negative-sense genomic RNA as a template, a complementary
+ssRNA is synthesized; this is then used as a template for the synthesis
of new genomic (-)ssRNA, which is rapidly encapsidated. Replication
presumably starts when enough nucleoprotein is present to encapsidate
neo-synthetized antigenomes and genomes.

Budding

The newly formed nucleocapsids and envelope proteins associate at the
host cell’s plasma membrane; budding occurs, destroying the cell.

These viruses recruit components of the cellular ESCRT (endosomal
sorting complex required for transport) system to mediate host-assisted
viral budding. SCRT complexes are normally used by the cell for
biological functions involving membrane remodeling, such as intra-luminal vesicle formation, autophagy or terminal stages of cytokinesis.
The ESCRT family consists of ESCRT-0, ESCRT-I, ESCRT-II which are
primarily involved in cargo sorting and membrane deformation, and
ESCRT-III which cleaves the bud neck from its cytosolic face.  In the
last step, vps4 disassembles the complex. The budding reaction
catalyzed by the ESCRT machinery has reversed topology when
compared with most other budding processes in the cell, such as
endocytosis and formation of transport vesicles.

Release –   Finally, the virion is released.

Favipiravir elicits antiviral mutagenesis during virus replication in vivo

Armando Arias, Lucy Thorne, Ian Goodfellow
eLife 2014;3:e03679. http://dx.doi.org:/10.7554/eLife.03679

Lethal mutagenesis has emerged as a novel potential therapeutic
approach to treat viral infections. Several studies have demonstrated
that increases in the high mutation rates inherent to RNA viruses lead
to viral extinction in cell culture, but evidence during infections in vivo
is limited. In this study, we show that the broad-range antiviral nucleoside
favipiravir reduces viral load in vivo by exerting antiviral mutagenesis
in a mouse model for norovirus infection. Increased mutation frequencies
were observed in samples from treated mice and were accompanied with
lower or in some cases undetectable levels of infectious virus in faeces
and tissues. Viral RNA isolated from treated animals showed reduced
infectivity, a feature of populations approaching extinction during antiviral
mutagenesis. These results suggest that favipiravir can induce norovirus
mutagenesis in vivo, which in some cases leads to virus extinction,
providing a proof-of-principle for the use of favipiravir derivatives or
mutagenic nucleosides in the clinical treatment of noroviruses.

Individualization of antiretroviral therapy
Rebecca Pavlos, Elizabeth J Phillips
Pharmacogenomics and Personalized Medicine 2012:5 1–17
http://dx.doi.org/10.2147/PGPM.S15303

Antiretroviral therapy (ART) has evolved considerably over the last
three decades. From the early days of monotherapy with high toxicities
and pill burdens, through to larger pill burdens and more potent
combination therapies, and finally, from 2005 and beyond where we
now have the choice of low pill burdens and once-daily therapies.
More convenient and less toxic regimens are also becoming available,
even in resource-poor settings. An understanding of the individual
variation in response to ART, both efficacy and toxicity, has evolved
over this time. The strong association of the major histocompatibility
class I allele HLA-B*5701 and abacavir hypersensitivity, and its
translation and use in routine HIV clinical practice as a predictive
marker with 100% negative predictive value, has been a success story
and a notable example of the challenges and triumphs in bringing
pharmacogenetics to the clinic. In real clinical practice, however, it is
going to be the exception rather than the rule that individual
biomarkers will definitively guide patient therapy. The need for
individualized approaches to ART has been further increased by the
importance of non-AIDS comorbidities in HIV clinical practice. In the
future, the ideal utilization of the individualized approach to ART will
likely consist of a combined approach using a combination of knowledge
of drug, virus, and host (pharmacogenetic and pharmacoecologic [factors
in the individual’s environment that may be dynamic over time])
information to guide the truly personalized prescription. This review
will focus on our knowledge of the pharmacogenetics of the efficacy
and toxicity of currently available antiretroviral agents and the current
and potential utility of such information and approaches in present
and future HIV clinical care.

Additional Considerations

PLOS Biology Paper Wins Omenn Prize for Viral Evasion Story
By Roli Roberts  Posted: June 19, 2014

We talk to the authors of a PLOS Biology research article published in
May 2013 that won the Omenn Prize for the best article published in 2013.
The Omenn Prize is awarded annually by the Evolution, Medicine, & Public
Health Foundation to authors of articles related to “evolution in the context
of medicine and public health,” and the winner was picked from a tough
long-list of 47 papers. Four other papers, including two from our sister
journal PLOS Pathogens, were cited for “honorable mention”
(Graves et al. Huijben et al.).

The PLOS Biology paper looks at how an essential mammalian protein –
the transferrin receptor, TfR1 – evolves in the face of contrasting selective
pressures. TfR1 is a protein that sits on the membrane of our cells and
mediates the regulated uptake of iron. TfR1 is stuck in the horns of a
dilemma. On the one hand, it has to be able to bind its functional partners –
the iron-loaded plasma protein transferrin, and a negative regulator protein
called HFE; this requirement constrains the sequence and structure of TfR1
through evolutionary time. On the other hand, it has to evade viruses that
exploit its handy cell-surface location, such as arenaviruses and the rodent
retrovirus MMTV.

TfR1 can carry on binding transferrin and HFE while dodging viruses

TfR1 can carry on binding transferrin and HFE while dodging viruses

TfR1 can carry on binding transferrin and HFE while dodging viruses.
http://dx.doi.org:/10.1371/journal.pbio.1001571

The authors compared sequences of TfR1 from various mammalian hosts
and then expressed them on the surface of cells to check a) their ability
to confer vulnerability or resistance to MMTV and arenaviruses such as
Machupo, Junin and Guanarito virus and b) their ability to bind to
transferrin. This image from the paper summarises the central finding –
how TfR1 (green) manages to square this circle by evolving rapidly (red)
to change the outer surfaces that are hijacked by viruses while keeping
constant the central surfaces that it uses to bind transferrin and HFE
(purple, blue).

First author Ann Demogines and lead author Sara Sawyer – both from
the University of Texas at Austin – told us how the study first arose and
then evolved into the paper that you can now read on our website.

Sawyer recalls the exact point at which the project started: “In the first year
of my faculty position, Welkin Johnson invited me to give a talk at the New
England Primate Research Center.  While I was there, I had a 45 minute
meeting with his colleague, Mike Farzan [also a co-author]. Mike had just
discovered TfR1 as the cellular receptor for arenaviruses, and suggested to
me that this might be a molecule that is engaged in an evolutionary arms race.
While ideas like this often arise out of conversations between scientists, I
remember having a gut reaction that this was something worth pursuing.”

Positively selected amino acids (red) hit the virus binding sites on TfR1 (blue, grey).

Positively selected amino acids (red) hit the virus binding sites on TfR1 (blue, grey).

Positively selected amino acids (red) hit the virus binding sites on
TfR1 (blue, grey)   http://dx.doi.org:/10.1371/journal.pbio.1001571

Demogines, who received $5000 from the Foundation, describes
how the spectacular arrangement of the evolutionarily selected sites
emerged: “I am still amazed to this day by the results of the evolutionary
analysis.  We were able to take DNA sequence from just 7 species and
computationally predict six sites under selection.  These sites were
scattered on the linear diagram of the protein, and didn’t make much
sense to us. But, when we placed them onto the 3D crystal structure
they formed a beautiful ridge going straight down the outer surface of
the receptor.  That was a great day in the lab.  We knew this had to
mean something!”

Demogines goes on to think about the implications of her paper and
related studies: “This work really gets me excited about the future of
evolutionary analysis applied in biomedical research.  As we collect the
genome sequences from more and more species, especially rodents and
bats which are major reservoirs for zoonotic and potentially zoonotic
viruses, we should be able to do this type of analysis more and more.
This type of analysis has many applications: allowing us to identify
critical cofactors involved in the viral lifecycle, viral binding sites, and
potentially novel drug targets. It can also be used to study interactions
with bacterial pathogens, although this has not yet been extensively explored.”

If you’d like to find out more about this elegant study, why not read the
article itself, or the accompanying Primer written by John Coffin:

“Dual Host-Virus Arms Races Shape an Essential Housekeeping Protein”
by Ann Demogines, Jonathan Abraham, Hyeryun Choe, Michael Farzan and
Sara L. Sawyer. http://dx.doi.org:/10.1371/journal.pbio.1001571

“Virions at the Gates: Receptors and the Host–Virus Arms Race” by
John M. Coffin. http://dx.doi.org:/10.1371/journal.pbio.1001574
Angiomotin Functions in HIV-1 Assembly and Budding
G Mercenne, SL Alam, J Arii, MS Lalonde and WI Sundquist
eLife 2015; http://dx.doi.org:/10.7554/eLife.03778

Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 ubiquitin E3 ligase family to facilitate virus release. Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif, suggesting that NEDD4L may function through an adaptor protein. Here, we demonstrate that the cellular protein Angiomotin (AMOT) can bind both NEDD4L and HIV-1 Gag. HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT,depletion, whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release. Electron microscopic
analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle. Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding.

The DEAH-Box RNA Helicase DHX15 Activates NF-B and MAPK Signaling Downstream of MAVS During Antiviral Responses

K Mosallanejad, Y Sekine, S Ishikura-Kinoshita, K Kumagai, T Nagano, et al.
Sci. Signal., 29 Apr 2014;  7(323), p. ra40
http://dx.doi.org/10.1126/scisignal.2004841]

During infection with an RNA virus, the DExD/H-box RNA helicases RIG-I (retinoic acid–inducible gene I) and MDA5 (melanoma differentiation–associated gene 5) activate the interferon regulatory factor 3 (IRF3), nuclear factor B (NF-B), c-Jun amino-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways through an unknown mechanism involving the adaptor protein MAVS (mitochondrial antiviral signaling). We used a Drosophila misexpression screen to identify DEAH-box polypeptide 15 (DHX15) as an activator of the p38 MAPK pathway. Human DHX15 contributed to the activation of the NF-B, JNK, and p38 MAPK pathways, but not the IRF3 pathway, in response to the synthetic double-stranded RNA analog poly(I:C) (polyinosinic-polycytidylic acid), and DHX15 was required for optimal cytokine production in response to poly(I:C) and infection with RNA virus. DHX15 physically interacted with MAVS and mediated the MAVS-dependent activation of the NF-B and MAPK pathways. Furthermore, DHX15 was required for poly(I:C)- and RNA virus–dependent, MAVS-mediated apoptosis. Thus, our findings indicate that, in RIG-I–like receptor signaling, DHX15 specifically stimulates the NF-B and MAPK pathways downstream of MAVS and contributes to MAVS-mediated cytokine production and apoptosis.

Mitochondrial Membrane Potential Is Required for MAVS-Mediated Antiviral Signaling
T Koshiba, Kai Yasukawa, Y Yanagi, and Shun-ichiro Kawabata
Sci. Signal., 1 Feb 2011; 4(158), p. ra7
http://dx.doi.org:/10.1126/scisignal.2001147]

Mitochondria, dynamic organelles that undergo cycles of fusion and fission, are the powerhouses of eukaryotic cells and are also involved in cellular innate antiviral immunity in mammals. Mitochondrial antiviral immunity depends on activation of the cytoplasmic retinoic acid–inducible gene I (RIG-I)–like receptor (RLR) signaling pathway and the participation of a mitochondrial outer membrane adaptor protein called MAVS (mitochondrial antiviral signaling). We found that cells that lack the ability to undergo mitochondrial fusion as a result of targeted deletion of both mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) exhibited impaired induction of interferons and proinflammatory cytokines in response to viral infection, resulting in increased viral replication. In contrast, cells with null mutations in either Mfn1 or Mfn2 retained their RLR-induced antiviral responses. We also found that a reduced mitochondrial membrane potential (m) correlated with the reduced antiviral response. The dissipation in m did not affect the activation of the transcription factor interferon regulatory factor 3 downstream of MAVS, which suggests that m and MAVS are coupled at the same stage in the RLR signaling pathway. Our results provide evidence that the physiological function of mitochondria plays a key role in innate antiviral immunity.

The Ubiquitin-Specific Protease USP15 Promotes RIG-I–Mediated Antiviral Signaling by Deubiquitylating TRIM25
Eva-Katharina Pauli, YK Chan, ME Davis, S Gableske, MK Wang, KF F, et al.
Sci. Signal., 7 Jan 2014; 7(307), p. ra3
http://dx.doi.org:/10.1126/scisignal.2004577

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys63)–linked ubiquitin chains to the RNA sensor retinoic acid–inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys48-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys48-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I–dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I–mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

Antiviral Mitochondrial Action
Editor’s Summary
Sci. Signal., 1 Feb 2011;  4(158), p. ra7
http://dx.doi.org:/10.1126/scisignal.2001147

Mitochondria are the energy generators of the cell, but they also act as platforms upon which complexes of proteins respond to RNA-containing viruses within the cytosol. Through genetic and pharmacological means, Koshiba et al. present evidence that suggests that the contribution of mitochondria to these antiviral responses is not as passive as originally thought. Indeed, their data suggest that successful resistance to viral infections depends on maintenance of the internal physiological functions of mitochondria coupled with the functions of the external protein complexes.

Prolonging Antiviral Signaling
Editor’s Summary
Sci. Signal., 7 Jan 2014; 7(307), p. ra3
http://dx.doi.org:/10.1126/scisignal.2004577

As part of the innate immune response to infection by RNA viruses, signaling by the cytosolic RNA sensor retinoic acid–inducible gene-I (RIG-I) stimulates the production of type I interferons (IFNs). RIG-I activity depends on its Lys63-linked polyubiquitylation by the ubiquitin E3 ligase TRIM25. As part of a negative feedback mechanism, the linear ubiquitin assembly complex (LUBAC) mediates the Lys48-linked polyubiquitylation and degradation of TRIM25, leading to inhibition of RIG-I signaling. Pauli et al. found that the deubiquitylase USP15 (ubiquitin-specific protease 15) counteracted the function of LUBAC by removing polyubiquitin from TRIM25, thus promoting its stabilization and enhancing the RIG-I–dependent antiviral response. Loss of USP15 resulted in decreased type I IFN production and enhanced viral replication in infected cells. Together, these data suggest that USP15 fine-tunes the antiviral response by sustaining the production of IFNs.

NIH-funded study uncovers range of molecular alterations in head and neck cancers, new potential drug targets

TCGA tumor genome sequencing analyses offer new insights into the effects of HPV and smoking, and find genomic similarities with other cancers

human papillomavirus (HPV) in head and neck cancer

human papillomavirus (HPV) in head and neck cancer

TCGA researchers have uncovered new details about the potential role of the human papillomavirus (HPV) in head and neck cancer. HPV-related head and neck cancers have been growing in number

Bethesda, Md., Wed., Jan. 28, 2015 – Investigators with The Cancer Genome Atlas (TCGA) Research Network have discovered genomic differences – with potentially important clinical implications – in head and neck cancers caused by infection with the human papillomavirus (HPV). HPV is the most common sexually transmitted virus in the United States, and the number of HPV-related head and neck cancers has been growing. Almost every sexually active person will acquire HPV at some point in their lives, according to the Centers for Disease Control and Prevention.

The researchers also uncovered new smoking-related cancer subtypes and potential new drug targets, and found numerous genomic similarities with other cancer types. Taken together, this study’s findings may provide more detailed explanations of how HPV infection and smoking play roles in head and neck cancer risk and disease development, and offer potential novel diagnostic and treatment directions.

The study is the most comprehensive examination to date of genomic alterations in head and neck cancers. The results were published online Jan. 28, 2015 in the journal Nature. TCGA is jointly supported and managed by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), both parts of the National Institutes of Health.

The U.S. Food and Drug Administration-approved HPV vaccines should be able to prevent the cancers caused by HPV infection in head and neck cancers and elsewhere, including anal cancer, whose incidence has also been increasing.
However, these vaccines work by preventing new infections, and the long interval between infection and cancer development make it important to understand the molecular changes that bring about these HPV-positive head and neck cancers – as well as those that lead to the HPV-negative cancers – and to develop new approaches for treating them.

“The rapid increase in HPV-related head and neck cancers, noticeably in oropharyngeal tumors, has created an even greater sense of urgency in the field,” said D. Neil Hayes, M.D., M.P.H, senior author of the study report and associate professor of medicine at the University of North Carolina (UNC) and the UNC Lineberger Comprehensive Cancer Center at Chapel Hill. Oropharyngeal cancer starts in the oropharynx, which is the part of the throat just behind the mouth. “We’re uncovering differences between tumors with and without HPV infection, and these new data are allowing us to rethink how we approach head and neck cancers.”

In the study, researchers performed genomic analyses on 279 tumors – head and neck squamous cell carcinomas (HNSCC) – from untreated patients. Approximately 80 percent of tumor samples were from individuals who smoked. The majority of samples were oral cavity cancers (61 percent) and larynx cancers (26 percent).

While only about 25 percent of head and neck cancers are linked to HPV infection, TCGA researchers confirmed that many patients with HPV-associated tumors have specific alterations of the gene FGFR3 and mutations in the PIK3CA gene, which are also found in a much broader set of mutations in smoking-related tumors. In contrast, while the EGFR (epidermal growth factor receptor) gene is frequently altered in HPV-negative tumors in smokers, it is rarely abnormal in HPV-positive tumors. Such insights may help in developing potential therapies and biomarkers, noted Dr. Hayes.

Head and neck cancers comprise a constellation of tumors of the mouth, throat, larynx, nasal cavity, salivary gland and elsewhere that have frequently been attributed to tobacco and alcohol use in most patients. Some 90 percent are squamous cell carcinomas, which occur in the surface layers of cells in the body. An estimated 55,000 people developed head and neck cancer in the United States in 2014. Approximately 12,000 Americans die from the diseases each year. Head and neck cancers are common worldwide, with more than 600,000 cases diagnosed each year.

“The rising worldwide incidence of head and neck cancers makes these types of large integrated genomic analyses by TCGA vital to establish a more detailed understanding of disease causes and behavior, and for the development of new treatment approaches,” said NIH Director Francis S. Collins, M.D., Ph.D.

Scientists found that more than 70 percent of head and neck cancers had alterations in genes for growth factor receptors (EGFR, FGFR, IGFR, MET, ERBB2, DDR2), signaling molecules (PIK3CA, HRAS) and cell division regulation (CCND1). These genes may play roles in pathways that control cell growth and proliferation, and for which therapies are either available or in development.

The investigators also discovered new clues about drug resistance in head and neck cancers. They found that genes affecting about 40 percent of such cancers form key parts of a pathway that helps determine cell survival and drug resistance. They showed that extra copies of the genes FADD and BIRC2, or mutations in or the absence of the CASP8 gene in smoking-related cancers – all which affect the process of programmed cell death – may underlie the resistance of cancer cells to current treatments. Similarly, the absence of the TRAF3 gene, or extra copies of a gene for the growth-promoting E2F1 protein in HPV-related cancers, may also increase resistance.

The findings showed similarities between head and neck cancer genomes and other cancers, including squamous cell lung and cervical, indicating possible common paths of cancer development, and potential treatment opportunities. “It is surprising to see that head and neck tumor genomes are remarkably similar to cervical and squamous lung cancer genomes. They are from very different organs, but they show similar losses and gains of genetic material across tumors,” Dr. Hayes noted. These common genetic abnormalities belong to a pathway that protects cells from damage and stress.

“These novel findings help establish a genomic map of various head and neck cancers, provide new insights into other similar cancers and  may further our understanding of how viruses can impact disease,” said NHGRI Director Eric D. Green, M.D., Ph.D.

“While many head and neck cancers are preventable, they are increasingly common throughout the world, and often challenging to effectively treat over the long term,” said NCI Director Harold Varmus, M.D. “This type of broad analysis provides important new clues for future research and treatment directions.”

The TCGA Research Network has generated data and published analyses on a number of cancers, all of which can be found on the TCGA website, www.cancergenome.nih.gov

The TCGA Research Network consists of more than 150 researchers at dozens of institutions across the nation.  A list of participants is available at http://cancergenome.nih.gov/abouttcga/overview. More details about The Cancer Genome Atlas, including Quick Facts, Q&A, graphics, glossary, a brief guide to genomics and a media library of available images can be found at http://cancergenome.nih.gov.

NHGRI is one of the 27 institutes and centers at the National Institutes of Health. The NHGRI Extramural Research Program supports grants for research and training and career development at sites nationwide. Additional information about NHGRI can be found at http://www.genome.gov.

NCI leads the National Cancer Program and the NIH effort to dramatically reduce the burden of cancer and improve the lives of cancer patients and their families, through research into prevention and cancer biology, the development of new interventions, and the training and mentoring of new researchers. For more information about cancer, please visit the NCI website at http://www.cancer.gov or call NCI’s Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

The NIDCD supports and conducts research and research training on the normal and disordered processes of hearing, balance, taste, smell, voice, speech, and language and provides health information, based upon scientific discovery, to the public. For more information about NIDCD programs, see the NIDCD website.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 institutes and centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases.

INNATE IMMUNITY
Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation
Siqi Liu1, Xin Cai1, Jiaxi Wu1, Qian Cong2, Xiang Chen1,3, Tuo Li1, Fenghe Du1,3, Junyao Ren1, Youtong Wu1, Nick Grishin2,3, Zhijian J. Chen1,3,*
Science http://dx.doi.org:/10.1126/science.aaa2630

1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
3Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.

During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cyclic guanosine monophosphate–adenosine monophosphate synthase, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here, we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases inhibitor of nuclear factor κB subunit IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

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