Viruses: Friends, Foes, Change Agents

Cover In contrast to their negative reputation as disease causing agents, some viruses can perform crucial biological and evolutionary functions that help to shape the world we live in today, according to a new free report by the American Academy of Microbiology: Viruses Throughout Life and Time: Friends, Foes, Change Agents.

Viruses participate in essential Earth processes and influence all life forms on the planet, from contributing to biogeochemical cycles, shaping the atmospheric composition, and driving major speciation events. Recent metagenomic studies of viruses have indicated we know very little about the real world of viruses. Almost all published research is about the viruses that cause disease in humans and their domesticated plants and animals. This certainly represents only a very small fraction of the viruses that really exist. It is very important to understand the real world of viruses, as this can inform our basic understanding of life and its origins, as well as major earth phenomena like carbon cycling.

Beyond their pathogenic impact, the report examines in depth the size of the virosphere, the origin of viruses, the overlooked biological and microbial ecological role of viruses, and how these live forms have contributed to evolution. Additional highlights from the report explain how some viruses are commensal organisms or symbionts, their functioning in microbial communities, and their role in maintaining the biosphere. The array of responsibilities taken on by viruses is due to their incredible sequence diversity and genomic plasticity, referred to as “viral dark matter”.

The report concludes by stimulating the readers to think about key questions: “What if viruses had never existed on Earth? Would life have evolved quite differently?” Continued virus research will help to answer these enticing questions.


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Gene editing eradicates HIV-1

CRISPR More than three decades after the discovery of HIV, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy fails to eradicate HIV infection, partly due to the persistence of virus reservoirs. A new paper just published shows that RNA-guided HIV-1 genome cleavage by Cas9 CRISPR technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing virus gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants might be developed quickly.

Comment: In spite of the breezy optimism of this paper (and this is progress), the work described has only been carried out on cultured cells in vitro. It is not clear whether or how easily it will be to replicate this finding in animals, and we’re still along way away from clinical trials which will be needed to show if this approach works in HIV-infected people.


RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. PNAS USA July 21, 2014, doi: 10.1073/pnas.1405186111
AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

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Mosquitoes infected with Wolbachia more likely to transmit West Nile virus

Wolbachia Mosquitoes infected with the bacteria Wolbachia are more likely to become infected with West Nile virus and more likely to transmit the virus to humans, according to a new paper.

Previous research has shown that Wolbachia – a genus of bacteria that live in insects – renders mosquitoes resistant to pathogen infection, preventing the mosquitoes from infecting humans with the pathogens. As a result, researchers are currently releasing Wolbachia-infected mosquitoes into the wild as part of a strategy to control Dengue virus. They also are investigating Wolbachia as a possible control strategy for malaria.

Expecting to find that Wolbachia would block infection by West Nile virus in the same way that it blocks Dengue virus, injected the Wolbachia into adult female Culex tarsalis mosquitoes, allowed them to grow, then fed the mosquitoes a meal of blood infected with West Nile virus. Wolbachia infection did not block West Nile virus in the mosquito, instead these mosquitoes had significantly higher West Nile virus infection rates seven days after they were fed the infected blood. Wolbachia infection allowed the mosquitoes to become infected with West Nile virus faster than the controls.

These results point to a previously unforeseen complication – the possibility that mosquitoes rendered resistant to one pathogen by Wolbachia infection might become better vectors of an alternative pathogen. The team also found that West Nile virus enhancement in the Wolbachia-infected mosquitoes occurred in conjunction with the suppression of genes associated with the mosquitoes’ anti-viral immune response.

This is the first study to demonstrate that Wolbachia can enhance a human pathogen in a mosquito. The results suggest that caution should be used when releasing Wolbachia-infected mosquitoes into nature to control vector-borne diseases of humans.


Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis. (2014) PLoS Negl Trop Dis 8(7): e2965. doi:10.1371/journal.pntd.0002965
Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector- borne disease control program.


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Honing in on enteric fever

Salmonella typhimurium Enteric fever (typhoid), affects about 22 million people and causes about 200,000 deaths every year, according to conservative estimates. Enteric fever is spread by bacteria belonging to the Salmonella genus, with two sub-species – Salmonella Typhi and Salmonella Paratyphi A – being responsible for most cases of the disease. And although the number of cases of enteric fever has fallen significantly over recent decades, there is a clear need for a diagnostic test for Salmonella that is rapid, affordable and accurate. It is important to be able to distinguish between enteric fever caused by Salmonella Typhi and enteric fever caused by Salmonella Paratyphi A in order to ensure that the correct drugs are prescribed and to combat the development of antibiotic resistance.

The application of metabolomics is relatively new in infectious diseases research compared to the application of genomics and proteomics. Despite this, screening the metabolome in blood plasma has identified useful prognostic profiles of several diseases, including sepsis. One of the major benefits of this technique is that it utilizes a pattern of biomarkers (that is, the various metabolites), as opposed to relying on just one host biomarker, as has been the focus of previous approaches.

A new paper in eLife applies this promising new approach to this challenge. Instead of trying to detect Salmonella in the blood during infection, they used a technique called metabolomics. The basic idea of this approach is that infection leads to metabolic changes, such that a person with enteric fever (or any infection) could have a profile of metabolites in their blood that is different to the metabolite profile of a healthy person. The challenge, therefore, is to identify a ‘metabolic fingerprint’ that can be used to detect enteric fever with high levels of sensitivity and specificity.


eLife: Host-pathogen interactions: Honing in on enteric fever


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A synthetic antibody with broad antiviral activity

Antibody Most strategies for developing virus-resistant transgenic cells and animals are based on the concept of virus-derived resistance, in which dysfunctional virus-derived products are expressed to interfere with the pathogenic process of the virus in transgenic cells or animals. However, these viral protein targeting approaches are limited because they only target specific viruses and are susceptible to viral mutations.

A new paper describes a novel strategy that targets the viral genome rather than virus gene products to generate virus-resistant transgenic cells and animals. A synthetic mini antibody (3D8 scFv), which has both DNase and RNase activities, was expressed in HeLa cells and transgenic mice. The authours found that the transgenic cells and mice acquired complete resistance to two DNA viruses and showed delayed onset of disease symptoms.

The antiviral effects against DNA viruses demonstrated in this study were caused by (1) DNase activity of the antibody in the nucleus, which inhibited DNA replication or RNA transcription and (2) antibody RNase activity in the cytoplasm, which blocked protein translation. This strategy might allow control of a broad spectrum of viruses, including viruses uncharacterized at the molecular level, regardless of their genome type or variations in gene products.


A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice. (2014) PLOS Pathog 10(6): e1004208. doi:10.1371/journal.ppat.1004208
Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.


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Crossing the species barrier

The number of pathogens known to infect humans is ever increasing. Whether such increase reflects improved surveillance and detection or actual emergence of novel pathogens is unclear. Nonetheless, infectious diseases are the second leading cause of human mortality and disability-adjusted life years lost worldwide. On average, three to four new pathogen species are detected in the human population every year. Most of these emerging pathogens originate from nonhuman animal species.

Zoonotic pathogens represent approximately 60% of all known pathogens able to infect humans. Their occurrence in humans relies on the human-animal interface, defined as the continuum of contacts between humans and animals, their environments, or their products. The human-animal interface has existed since the first footsteps of the human species and its hominin ancestors 6–7 million years ago, promoting the prehistoric emergence of now well-established human pathogens. These presumably include pathogens with roles in the origin of chronic diseases, such as human T-lymphotropic viruses and Helicobacter pylori, as well as pathogens causing major crowd diseases, such as the smallpox and measles viruses and Bordetella pertussis. Since prehistory, the human-animal interface has continued to evolve and expand, ever allowing new pathogens to access the human host and cross species barriers.

Crossing the Interspecies Barrier: Opening the Door to Zoonotic Pathogens. (2014) PLoS Pathog 10(6): e1004129. doi:10.1371/journal.ppat.1004129

Crossing the species barrier

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In case you forgot – we're still fighting vCJD

Prions The first cases of Mad Cow disease in humans (properly called variant Creutzfeld Jakob Disease, vCJD) occurred in the late 1990s as the consequence of eating contaminated beef products. Since then, several cases of secondary infections caused by transfusions with blood from donors who subsequently developed vCJD have been reported, raising ongoing concerns about the safety of blood and blood products. A paper just published describes a new test that uses protein misfolding cyclic amplification (PMCA – like PCR for proteins) which can detect prions in blood samples from humans with vCJD and in animals at early stages of the (asymptomatic) incubation phase.

This test could be used to identify vCJD infected but asymptomatic individuals and/or for screening donated blood for the presence of the vCJD agent. In the UK, 1 out 2000 people could carry the vCJD agent. In the absence of a vCJD screen, the UK like most of the developed countries apply systematic measures aiming at mitigating the blood borne transmission risk of the disease. These measures have a substantial cost and increase the difficulty met by the blood banking system to provide certain blood products.


Preclinical Detection of Variant CJD and BSE Prions in Blood. (2014) PLoS Pathog 10(6):e1004202. doi: 10.1371/journal.ppat.1004202
The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.


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Virus ecology

Viruses Nothing wildly new here, but rather a nice overview of viruses from an ecological rather than a disease perspective.

What Ecologists Can Tell Virologists. Annual Review of Microbiology, first posted online on May 16, 2014. doi: 10.1146/annurev-micro-091313-103436

I pictured myself as a virus…and tried to sense what it would be like. — Jonas Salk

Ecology as a science evolved from natural history, the observational study of the interactions of plants and animals with each other and their environments. As natural history matured, it became increasingly quantitative, experimental, and taxonomically broad. Focus diversified beyond the Eukarya to include the hidden world of microbial life. Microbes, particularly viruses, were shown to exist in unfathomable numbers, affecting every living organism. Slowly viruses came to be viewed in an ecological context rather than as abstract, disease-causing agents. This shift is exemplified by an increasing tendency to refer to viruses as living organisms instead of inert particles. In recent years, researchers have recognized the critical contributions of viruses to fundamental ecological processes such as biogeochemical cycling, competition, community structuring, and horizontal gene transfer. This review describes virus ecology from a virus’s perspective. If we are, like Jonas Salk, to imagine ourselves as a virus, what kind of world would we experience?



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A new drug against MERS?

MERS virus Before the emergence of the highly pathogenic severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2003 only two circulating human coronaviruses (HCoVs), HCoV- 229E and HCoV-OC43 causing relatively mild common cold-like respiratory tract infections, were known, and coronaviruses have not been regarded as significant threat for human health. Now, more than ten years later, the emergence of another highly pathogenic coronavirus of zoonotic origin, the Middle East respiratory syndrome coronavirus (MERS-CoV) points to the need for effective drugs against coronaviruses. Viruses such as coronaviruses that replicate in the host cell cytoplasm have evolved to employ host cell-derived membranes to compartmentalize genome replication and transcription. Specifically for positive-stranded RNA viruses, accumulating knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis specify the establishment of the viral replicase complex at host cell-derived membranes as an evolution- ary conserved and essential step in the early phase of the viral life cycle.

A new paper in PLoS Pathogens describe a small molecule inhibitor of coronavirus replication that specifically targets this membrane-bound RNA replication step and has broad antiviral activity against number of diverse coronaviruses including highly pathogenic SARS-CoV and MERS-CoV. Since resistance mutations appear in an integral membrane-spanning component of the coronavirus replicase complex, and since all positive stranded RNA viruses have very similar membrane-spanning or membrane-associated replicase components implicated in anchoring the viral replication complex to host cell-derived membranes, the data suggests that the membrane-bound replication step of the viral life cycle is a novel, vulnerable, and druggable target for antiviral intervention of a wide range of RNA virus infections.

Of course clinical trials are needed before such drugs could be used, so we’re still years away from this approach being put into practce. Just in time for the next emergent coronavirus maybe?


Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus. (2014) PLoS Pathog 10(5): e1004166. doi:10.1371/journal.ppat.1004166
Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.


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