Solving the mystery of reverse transcription

Cytoplasmic reverse-transcription and pre-integration complexes The biology of retroviruses is really all about the processes of reverse transcription, so it’s frustrating that nearly 50 years after their discovery, the steps from cytoplasmic entry until integration of the reverse transcribed genome are still mysterious. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated.

A new paper in eLife describes quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. Interestingly, the virus capsid (CA) protein remains associated with cytoplasmic RTC/PIC in primary human macrophages, but is lost from nuclear PIC in a HeLa-derived cell line. The capsid-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. This newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.

Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid. eLife December 17, 2014 doi: 10.7554/eLife.04114

 

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Human Viruses and Cancer

The first human tumor virus was discovered in the middle of the last century by Anthony Epstein, Bert Achong and Yvonne Barr in African pediatric patients with Burkitt’s lymphoma. To date, seven viruses – EBV, KSHV, high-risk HPV, MCPV, HBV, HCV and HTLV – have been consistently linked to different types of human cancer, and infections are estimated to account for up to 20% of all cancer cases worldwide. Viral oncogenic mechanisms generally include: generation of genomic instability, increase in the rate of cell proliferation, resistance to apoptosis, alterations in DNA repair mechanisms and cell polarity changes, which often coexist with evasion mechanisms of the antiviral immune response. Viral agents also indirectly contribute to the development of cancer mainly through immunosuppression or chronic inflammation, but also through chronic antigenic stimulation. There is also evidence that viruses can modulate the malignant properties of an established tumor. In the present work, causation criteria for viruses and cancer will be described, as well as the viral agents that comply with these criteria in human tumors, their epidemiological and biological characteristics, the molecular mechanisms by which they induce cellular transformation and their associated cancers.

Morales-Sánchez, A., and Fuentes-Pananá, E.M. (2014) Human Viruses and Cancer. Viruses, 6(10): 4047-4079.

Bradford Hill's postulates for causative relations

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Oncolytic viruses as anticancer vaccines

Oncolytic viruses as anticancer vaccines
Oncolytic virotherapy has shown impressive results in preclinical studies and first promising therapeutic outcomes in clinical trials as well. Since viruses are known for a long time as excellent vaccination agents, oncolytic viruses are now designed as novel anticancer agents combining the aspect of lysis-dependent cytoreductive activity with concomitant induction of antitumoral immune responses. Antitumoral immune activation by oncolytic virus infection of tumor tissue comprises both, immediate effects of innate immunity and also adaptive responses for long lasting antitumoral activity, which is regarded as the most prominent challenge in clinical oncology. To date, the complex effects of a viral tumor infection on the tumor microenvironment and the consequences for the tumor-infiltrating immune cell compartment are poorly understood. However, there is more and more evidence that a tumor infection by an oncolytic virus opens up a number of options for further immunomodulating interventions such as systemic chemotherapy, generic immunostimulating strategies, dendritic cell-based vaccines, and antigenic libraries to further support clinical efficacy of oncolytic virotherapy.
Oncolytic viruses as anticancer vaccines. (2014) Front Oncol. 4:188. doi: 10.3389/fonc.2014.00188

 

Oncolytic viruses are natural or genetically modified viral species that selectively infect and kill neoplastic cells. Such an innate or exogenously conferred specificity has generated considerable interest around the possibility to employ oncolytic viruses as highly targeted agents that would mediate cancer cell-autonomous anticancer effects. Accumulating evidence, however, suggests that the therapeutic potential of oncolytic virotherapy is not a simple consequence of the cytopathic effect, but strongly relies on the induction of an endogenous immune response against transformed cells. In line with this notion, superior anticancer effects are being observed when oncolytic viruses are engineered to express (or co-administered with) immunostimulatory molecules. Although multiple studies have shown that oncolytic viruses are well tolerated by cancer patients, the full-blown therapeutic potential of oncolytic virotherapy, especially when implemented in the absence of immunostimulatory interventions, remains unclear. Here, we cover the latest advances in this active area of translational investigation, summarizing high-impact studies that have been published during the last 12 months and discussing clinical trials that have been initiated in the same period to assess the therapeutic potential of oncolytic virotherapy in oncological indications.

Trial Watch: Oncolytic viruses for cancer therapy. Oncoimmunology. 2014 3: e28694. doi: 10.4161/onci.28694

 

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Garlic for the common cold

Garlic I’ve been suffering with a cold for the past week so I was quite interested to see this updated metastudy from the Cochrane Database. I’m not convinced garlic can cure the comon cold, but I certainly haven’t been troubled by vampires recently. But on another note: what the heck is “the common cold” exactly, and what are these trials testing?

Listen to the podcast here:

Lissiman E, Bhasale AL, Cohen M. Garlic for the common cold. Cochrane Database of Systematic Reviews 2014, Issue 11. Art. No.: CD006206. DOI: 10.1002/14651858.CD006206.pub4
Background: Garlic is popularly believed to be useful for the common cold. This belief is based on traditional use and some laboratory evidence that garlic has antibacterial and antiviral properties. On average, adults have two to four common colds per year.
Study characteristics: The evidence is current to the 7 August 2014. Of the eight studies identified, only one fulfilled the criteria for the review. This study assessed 146 participants over a three-month period. Half the participants took a placebo tablet and half took a garlic tablet during this time. The participants then wrote in a diary when they had symptoms of a cold.
Key results: The included study found that people who took garlic every day for three months (instead of a placebo) had fewer colds. That is, over the three-month period, there were 24 occurrences of the common cold in the garlic group, compared to 65 in the placebo group. When participants experienced a cold, the length of illness was similar in both groups (4.63 versus 5.63 days).
Quality of the evidence: More participants in the garlic group (four) than the placebo group (one) noted a smell when burping, so it is possible that blinding of participants was not adequate. However, other potential biases were well controlled. The only included study is directly relevant to the review question. Although the trial is small, there were enough participants to provide precise, reliable results. There is no evidence that results were selectively reported. However, this was possible as the outcomes do not appear to have been decided in advance. Considering the financial incentive for supplement companies to produce positive trials, it is also possible that trials that showed no effect of garlic were never published. Overall, the quality of the evidence is moderate.
Side effects: Possible side effects in this small trial included odour and a skin rash. More information is needed about the possible side effects of garlic.

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The threat of zoonotic diseases

Crossing the species barrier Zoonotic infectious diseases have been important concerns to humans since the beginning of the domestication of animals 10,000 years ago. Approximately 75% of emerging infectious diseases are zoonoses. The phenomenon of emerging and reemerging infectious diseases is driven by various anthropogenic factors, including: genetic and biological factors, such as microbial adaptation to macro- and microenvironmental changes along with changes in host susceptibility to infection; environmental factors, including climate change, changes in ecosystems, and changes in human and animal population densities; and socioeconomic and political factors, such as increasing international travel and commerce, social inequality, poverty, conflict, famine, lack of political will, and changes in economic development and land use.

Over the last 15 years, our planet has faced more than 15 deadly zoonotic or vector-borne global outbreaks, both viral (e.g., Ebola, Hanta, highly pathogenic avian influenza [H5N1 and H7N9], West Nile, Rift Valley fever, norovirus, severe acute respiratory syndrome [SARS], Marburg, influenza A [H1N1]) and bacterial (e.g. Escherichia coli O157:H7, Yersinia pestis, and Bacillus anthracis). Since 1980, more than 87 new zoonotic and/or vector-borne EIDs have been discovered.

The global economic burden due to zoonotic diseases is very high. According to a recent World Bank estimate, the economic burden due to six of the zoonotic diseases that have occurred in specific countries between 1997 and 2009 is estimated to be US$80,000,000,000. In a worst-case scenario, potential losses from a pandemic influenza outbreak could be US$3 trillion, which is equivalent to 5% of the global GDP. A recent report from the International Livestock Research Institute highlighted zoonoses as major obstacles to poverty alleviation, affecting 1,000,000,000 livestock keepers. The report estimated that there are over 2,500,000,000 cases of human illness and 2.7 million deaths annually due to the top 56 zoonoses.

While zoonotic EIDs are a major concern globally, their impact in less developed countries is disproportionately high because of the occurrence of risk factors such as a high rate of population growth, lack of infrastructure and skilled-manpower capacity to tackle disease outbreaks, a high proportion of people with compromised immunity due to comorbidities such as HIV/AIDS or parasitic diseases, and lifestyles in which daily life depends on animals.

The Global One Health Paradigm: Challenges and Opportunities for Tackling Infectious Diseases at the Human, Animal, and Environment Interface in Low-Resource Settings. (2014) PLoS Negl Trop Dis 8(11): e3257. doi:10.1371/journal.pntd.0003257
Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.

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Small Is Beautiful

Influenza M2 Small Proteins Can No Longer Be Ignored. (2014) Annual Review of Biochemistry 83: 753-777 doi: 10.1146/annurev-biochem-070611-102400
Small proteins, here defined as proteins of 50 amino acids or fewer in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years, however, increasing numbers of small proteins have been identified either through the realization that mutations in intergenic regions are actually within unannotated small protein genes or through the discovery that some small, regulatory RNAs encode small proteins. These insights, together with comparative sequence analysis, indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review summarizes what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which these small proteins act and suggesting several general conclusions. Important questions for future studies of these overlooked proteins are also discussed.

Viral miniproteins. Annual Review of Microbiology (2014) 68: 21-43. doi: 10.1146/annurev-micro-091313-103727
Many viruses encode short transmembrane proteins that play vital roles in virus replication or virulence. Because many of these proteins are less than 50 amino acids long and not homologous to cellular proteins, their open reading frames were often overlooked during the initial annotation of viral genomes. Some of these proteins oligomerize in membranes and form ion channels. Other miniproteins bind to cellular transmembrane proteins and modulate their activity, whereas still others have an unknown mechanism of action. Based on the underlying principles of transmembrane miniprotein structure, it is possible to build artificial small transmembrane proteins that modulate a variety of biological processes. These findings suggest that short transmembrane proteins provide a versatile mechanism to regulate a wide range of cellular activities, and we speculate that cells also express many similar proteins that have not yet been discovered.

 

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Understanding the next Ebola virus rather than panicking about the current one

Parvovirus Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. This is an important example of a viral pathogen that evolved by cross-species transmission and mutation to initiate a disease pandemic, something virologists have been writing about for years but is now highly fashionable (think: Ebola: Will ebola come to the UK? Yes. And will it kill us all? No).

Carnivore parvoviruses infect many species, and their passage in different hosts may select mutations that facilitate host jumping; for example, natural passage of CPV in raccoons may have facilitated its adaptation to dogs. A new papers demonstrates that these barriers can be overcome by only a few mutations in the virus that probably alter host receptor binding, and that host adaptation can differ dramatically among very similar viruses.

Passage of viruses in cell cultures of different hosts results in mutations at the same sites that vary in nature and confer fitness increases, strongly suggesting that they are adaptively important. This shows that parvoviruses may cross species barriers to infect less susceptible hosts through single or only a few mutations, and that differences in the genetic background, host range, and/or evolutionary history of the viruses influence their propensity to jump hosts. These discoveries help reveal the mechanisms that control host switching and virus emergence.

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species. (2014) PLoS Pathog 10(11): e1004475. doi:10.1371/journal.ppat.1004475
Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of virus sequences recovered from different wild carnivore species, as shown here, demonstrated that 95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.

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Let’s get things in perspective

Chromobacterium While the current outbreak of Ebola virus in West Africa is very serious, in the current panic people have forgotten that malaria kills thousands of times more people than Ebola each year. In spite of decades of effort, we are still struggling with malaria vaccines, so attention is starting to switch to a new strategy, targeting the mosquitoes that spread malaria.

Just like those of humans, insect guts are full of microbes, and this microbiota can influence the insect’s ability to transmit diseases. A new study reports that a bacterium (Chromobacterium Csp_P) isolated from the gut of an Aedes mosquito can reduce infection of mosquitoes by malaria parasites and dengue virus. The bacterium can also directly inhibit these pathogens in the test tube, and shorten the life span of the mosquitoes that transmit both diseases.

Chromobacterium Csp_P can effectively colonize the midguts of Anopheles gambiae and Aedes aegypti mosquitoes and can, when ingested by the mosquito, significantly reduce the mosquito’s susceptibility to infection with the malaria parasite and dengue virus. We also show that exposure to, and ingestion of, Csp_P can reduce the lifespan of larval and adult mosquitoes. Csp_P has anti-Plasmodium and anti-dengue activity independent of the mosquito, suggesting that the bacterium secretes metabolites that could potentially be exploited to prevent disease transmission or to treat infection.

This comes on top of earlier work involving the use of Wolbachia to attack mosquitoes. We may be some way away from an effective malaria vaccine, but the development of novel control strategies for vector-borne diseases is gaining ground rapidly.

 

Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities. (2014) PLoS Pathog 10(10): e1004398. doi: 10.1371/journal.ppat.1004398
Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria- produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito’s vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P-produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.

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How to stop the spread of Ebola [video]

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