MicrobiologyBytes

New research shows that Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV cell entry factor that functions after binding, at or before fusion. Together with the facts that NPC1L1 is a cellular cholesterol receptor, the HCV particle is enriched in cholesterol, and relative dependence on NPC1L1 is correlated with HCV particle cholesterol levels, supports and expands on previous reports suggesting that virion cholesterol is involved in HCV cell entry. Whether NPC1L1 directly interacts with HCV or indirectly participates in HCV entry by removing virion-associated cholesterol to perhaps reveal protected viral glycoprotein binding sites or confer a required conformational change remains to be determined. As NPC1L1 is expressed only on human and primate hepatocytes, this discovery additionally highlights NPC1L1 as a potential HCV tropism determinant, which may facilitate the future development of animal models of HCV infection.

Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nature Medicine 08 January 2012 doi:10.1038/nm.2581
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture–derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol–dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

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Arenaviruses comprise a diverse family of enveloped negative-strand RNA viruses that are endemic to specific rodent hosts worldwide. Several arenaviruses cause severe hemorrhagic fevers in humans, including Junín and Machupo viruses in South America and Lassa fever virus in western Africa.

Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins.

This review describes the properties of the tripartite GPC complex and the evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention.

The Curious Case of Arenavirus Entry, and Its Inhibition. (2012) Viruses 4(1), 83-101 doi:10.3390/v4010083

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At the 64th gathering of the World Health Assembly in May 2011, a majority of delegates reaffirmed the view that the remaining stocks of variola virus, the causative agent of smallpox, should be destroyed. But consideration of precisely when that action should be taken was postponed for several years pending completion of crucial research addressing the development and assessment of tools for the treatment and control of the disease should it ever reoccur. This agenda of research includes the development and testing of vaccines and therapeutics, at the heart of which involves understanding the mechanisms that govern variola’s virulence and its capability to evade host defenses. But how can these questions be addressed when naturally occurring disease has been eliminated and no satisfactory animal model exists?

In the absence of smallpox, human infections with monkeypox virus constitute the most significant communicable Orthopoxvirus-associated illnesses extant in the world today. Monkeypox is not a direct research-proxy or substitute for smallpox (monkeypox is a zoonosis that can affect a broad range of animal taxa, whereas smallpox was an exclusive disease of humans) but vaccines and therapies developed for smallpox may be useful for the prevention and treatment of monkeypox, and many of the cornerstones of pathogenesis and immunomodulation for both variola and monkeypox probably emanate from conserved homologous processes. Both variola and monkeypox viruses are considered threat agents that could be used unlawfully for acts of bioterrorism. With monkeypox, there is the added question of its persistence in nature, and its potential for expansion in a world that is now more than 30 years without smallpox. A significant expansion of monkeypox in the world today could pose many of the same challenges as a resurgence of smallpox.

Outbreaks of human monkeypox after cessation of smallpox vaccination. Trends Microbiol. 10 Jan 2012
The recent observation of a surge in human monkeypox in the Democratic Republic of the Congo (DRC) prompts the question of whether cessation of smallpox vaccination is driving the phenomenon, and if so, why is re-emergence not universal throughout the historic geographic range of the virus? Research addressing the virus’s mechanisms for immune evasion and induction, as well as that directed at elucidating the genes involved in pathogenesis in different viral lineages (West African vs Congo Basin), provide insights to help explain why emergence appears to be geographically limited. Novel vaccines offer one solution to curtail the spread of this disease.

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Herpes simplex virus (HSV) is an important pathogenic agent that causes recurrent oral and genital lesions, blindness and encephalitis. It is a member of the family Herpesviridae, which contains three subfamilies (alpha- beta- and gammaherpesvirinae) whose members infect humans to cause a variety of ailments, from benign rashes to nasopharyngeal carcinoma. Although this review focuses on HSV, the assembly steps that occur in the nucleus and the proteins involved are highly conserved among all family members, which suggests that antiviral agents that block these steps might be effective against many different herpesviruses and their associated diseases. Despite this potential, a broadly effective compound has yet to be realized, in part because many of the processes are only poorly understood in sufficient molecular detail. This review outlines these intranuclear assembly steps and illustrate potential and existing antiviral strategies that exploit them.

Herpes simplex virus capsid assembly and DNA packaging: a present and future antiviral drug target. (2011) Trends Microbiol. 19(12) :606-613

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Recent evidence shows that the quadrivalent HPV vaccine prevents several HPV-related diseases in men. However, despite the licensure of the vaccine in the USA for men 9 through 26 years of age, rates of male vaccination are very low. Research on acceptability, in general, indicates strong interest in vaccination among men, parents, and healthcare providers, though female vaccination is typically seen as a higher priority. Cost-effectiveness studies indicate that in the context of modest female vaccination rates and with the specification of a broad range of disease outcomes (e.g. genital warts, anogenital cancers, and oropharyngeal cancers), male vaccination can be quite cost-effective.

This review describes the indications for vaccinating men with the quadrivalent human papillomavirus (HPV) vaccine, reports on the U.S. rates of male vaccination, and reviews the recent research on acceptability of vaccinating men and research on the cost-effectiveness of adding men to existing female HPV immunization programs.

Summary: Men are at high risk for HPV infection and can benefit from vaccination, but vaccination rates among men remain extremely low. More research needs to be done on the predictors of uptake of HPV vaccine among men and on the development of interventions to increase male vaccination.

Human papillomavirus vaccine and men: what are the obstacles and challenges? (2012) Curr Opin Infect Dis. 25(1): 86-91

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By convention, the human adenovirus replication cycle is divided into two phases, an early and a late phase, which are separated by the onset of viral DNA replication. Based on temporal changes of the gene expression pattern as revealed by DNA microarray analysis, adenovirus type 2 (Ad2) infection in human primary lung fibroblasts can be divided into four periods. The first period is from 0 to 12 h after infection before or shortly after adenoviral gene expression has commenced. During this time, changes in cellular gene expression are likely to be triggered by the virus entry process, such as attachment of virus to cell surface receptors, and its intracellular transport along microtubules.

The second period covers the time from 12 to 24 h after infection and follows activation of the immediate early E1A gene. During this period, there is an increase in the number of differentially expressed cellular genes. About 50% of these genes are involved in cell cycle regulation, cell proliferation and antiviral response. The third period extends from 24 to 42 h after infection. By this time, the virus has gained control of the cellular metabolic machinery, resulting in an efficient replication of the viral genome. Additional changes in cellular gene expression are modest during this phase. During the fourth and last period, when the cytopathic effect becomes apparent, the number of down-regulated genes increases dramatically including many genes involved in intra- and extracellular structure.

The most intensive battle between the adenovirus and its host takes place during the second period after adenovirus genes expression has started. The major functions of the early gene products are to force the host cell to enter the S phase in order to provide optimal conditions for viral DNA replication and to suppress the host antiviral response. Adenoviruses encode several regulatory proteins within the early regions E1A, E1B, E3, and E4. The immediate-early E1A gene encodes two regulators of viral and cellular gene expression, the E1A-243R and E1A-289R proteins. The E1A proteins act as promiscuous transcriptional activators or repressors of cellular genes. E1A proteins are essential for promoting the host cell to enter the S phase. This is achieved by the binding of the E1A proteins to members of the retinoblastoma tumor suppressor (pRB) family, thereby releasing the E2F transcription factors, which are activators of genes required in the S-phase.

The transcriptome of the adenovirus infected cell. Virology. 9 Jan 2012
Alternations of cellular gene expression following an adenovirus type 2 infection of human primary cells were studied by using superior sensitive cDNA sequencing. In total, 3791 cellular genes were identified as differentially expressed more than 2-fold. Genes involved in DNA replication, RNA transcription and cell cycle regulation were very abundant among the up-regulated genes. On the other hand, genes involved in various signaling pathways including TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB stood out among the down-regulated genes. Binding sites for E2F, ATF/CREB and AP2 were prevalent in the up-regulated genes, whereas binding sites for SRF and NF-κB were dominant among the down-regulated genes. It is evident that the adenovirus has gained a control of the host cell cycle, growth, immune response and apoptosis at 24h after infection. However, efforts from host cell to block the cell cycle progression and activate an antiviral response were also observed.

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When a virus infects a cell, it hijacks resources of the cell to manufacture and release a new generation of progeny virus particles. Yet despite its central importance, methods to precisely quantify virus production at the cellular level are lacking. Most approaches measure the production of virus by sampling from a population of infected cells, providing average values that mask the potentially wide-ranging and significant behaviors of individual cells.

Within a laboratory culture of virus-infected cells, or within a tissue of an infected host, individual cells can diverge in behavior from the average or majority of infected cells. However, these rare cells may nevertheless contribute importantly to the long-term behavior of the infection, well beyond their initial encounter with the virus. Because these rare cell behaviors are generally obscured during average-cell measures of infection, they highlight the need for single-cell measures of behavior that can be readily performed on many individual cells to reveal the extent of cell heterogeneity. This paper demonstrates a method to measure the kinetics of virus production from individual cells, without confounding effects from secondary infections. The method combines a series of simple steps that can be performed in any cell biology or virology facility, without reliance on specialized equipment.

Kinetics of virus production from single cells. Virology. 03 Jan 2012
The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yield spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

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First it was foot and mouth virus.
Then it was bluetongue virus.
Now it is Schmallenberg virus.
So here’s 10 things you didn’t know about Schmallenberg virus:

1. Schmallenberg virus was first isolated in Schmallenberg, Germany, in November 2011.
2. Schmallenberg virus is a Bunyavirus, one of a large group of of negative-stranded RNA viruses.
3. Why should I care? In cows, Schmallenberg virus causes fever and a drastic reduction in milk production. In sheep it causes congenital malformations and stillborn lambs (also stillborn calves in cows).
4. Schmallenberg virus was first identifed in the UK on 23rd January 2012.
5. Like Bluetongue, Schmallenberg virus is transmitted by midges (Culicoides spp.), which means we will be unlikely to be able to eradicate it – vaccination of anaimals is the only likely effective response.
6. Where did Schmallenberg virus come from? The virus genome is most closely related to sequences of a different Orthobunyavirus called Shamonda virus which belongs to the so-called Simbu serogroup known to infect ruminants and be transmitted by midges. In other words, it has form. But whether it is newly evolved (unlikely) or just newly discovered we don’t yet know.
7. How did Schmallenberg virus reach the UK? We don’t know. It could have been due to animal movements, but since it was first identifed in eastern England, it’s possible that it arrived in midges travelling under their own steam.
8. Is Schmallenberg virus going to spread to other parts of the UK and other countries? Yes, you can bet on that (just like bluetongue did).
9. Can I catch Schmallenberg virus? Honest answer: We don’t know. Possibly, but there have been no reports of human illness from areas where the virus is known to exist, so I wouldn’t worry too much.
10. Where can I find the latest news about Schmallenberg virus? Right here.
11. OK, one last time, why should I care? Because Schmallenberg virus is going to cost European and probably worldwide ecomonies millions of pounds. And that will affect you.

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Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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