MicrobiologyBytes

The main impediment to a cure for HIV is the existence of long-lasting treatment resistant virus reservoirs. This review discusses what is currently known about reservoirs, including their formation and maintenance, while focusing on latently infected CD4+ T cells. It compares several different in vivo and in vitro models of latency and comments on how each model may reflect the properties of reservoirs in vivo, especially with regard to cell phenotype, since recent studies demonstrate that multiple CD4+ T cell subsets contribute to HIV reservoirs and that with HAART and disease progression the relative contribution of different subsets may change. It also focuses on the direct infection of resting CD4+ T cells as a source of reservoir formation and as a model of latency, since recent results help explain the misconception that resting CD4+ T cells appeared to be resistant to HIV in vitro.

HIV reservoirs and latency models. Virology. 2011 411(2): 344-354

The complement system functions as an immune surveillance system that rapidly responds to infection. Activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. However, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. This review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection.

Complement and viral pathogenesis. Virology. 2011 411(2): 362-373

The most amazing thing about the “common cold”, or at least, the proportion of this spectrum of diseases which is caused by rhinoviruses (as opposed to adenoviruses, coronaviruses, or something else), is how little we still know about it. A few years ago I was involved in a proposal for a large project which would look at some of the issues adressed by a new research paper which has just appeared.  That proposal eventually collapsed under its own weight, so it’s fascinating to read these new results and see what we might have found.

The new study describes a phylogenetic analysis to compare the relative distribution of HRV species or serotypes according to the respiratory site (upper respiratory tract (URT) versus lower respiratory tract (LRT) ) and in protracted infection in hospital patients and immunosuppressed lung transplant recipients. In one case, rhinovirus genome variation was followed in the URT and LRT over a period of 27 months using both classical and ultra-deep sequencing methods.

Based on phylogenetic analysis, the frequency distribution of strains infecting the URT and LRT did not reveal any apparent correlation between a given HRV serotype or species and their ability to infect the LRT. Five lung transplant recipients were chronically infected with HRV during periods of time ranging from three to 27 months. Mutation mapping along the HRV genome showed that synonymous changes were roughly spread along the entire ORF, whereas non-synonymous changes clustered mostly in the capsid VP2, VP3, and VP1 genes. The capsid genes are also the most variable during acute infections in immunocompetent hosts.

As expected, the data suggests that immunocompromised patients cannot clear virus infections as well as immunocompetent individuals, and represent a potential reservoir for the emergence of new variants and inter-host transmission due to chronic virus infection. In addition, these patients may be co-infected by two viruses, thus opening the door to recombination, another putative driving force of rhinovirus evolution. With the emergence of new therapies and progress in transplantation, the population of immunocompromised patients is constantly increasing. Our results suggest that this could accelerate the ability of viruses to adapt to the host, evolve, and propagate and may favor a more rapid emergence of new viral variants.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections. (2011) PLoS ONE 6(6): e21163. doi:10.1371/journal.pone.0021163
Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5′ UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

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Two interesting recent papers:

When a pathogen attacks, the immune system rapidly mobilizes host defenses in order to reduce the microbial burden and limit damage to the host. Innate immunity is the first line of defense and relies on germ line–encoded pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs), which sense microbial products that are not normally found on or in mammalian cells. The considerable potency of nucleic acids as triggers of the innate immune response has gained appreciation over the last few years. In particular, nucleic acid sensing of viruses is central to anti-viral defenses through recognition of viral genomes or nucleic acids generated during viral replication. Distinct classes of nucleic acid sensing molecules have been uncovered that function in different cell types and subcellular compartments to coordinate innate defenses. While recognition of RNA molecules is dependent on members of the TLR family and cytosolic RNA helicases, the mechanisms underlying the sensing of DNA have been less well defined. It has been known for over a decade that DNA, the most recognizable unit of life, is a potent trigger of inflammatory responses in cells. The discovery of TLR-9, a receptor for hypomethylated CpG-rich DNA, partially explained these findings. TLR9 is localized to the endosomal compartment and in humans is expressed in B cells as well as in plasmacytoid dendritic cells (pDCs). However, it became clear that the immune stimulatory activity of microbial DNA was not compromised in many cells lacking TLR9. These observations prompted new efforts to understand how DNA triggers immune responses, an endeavor that has led to the discovery of several new DNA recognition receptors and fresh insights into infectious as well as autoimmune diseases:
Innate Immune Sensing of DNA. (2011)PLoS Pathog 7(4): e1001310. doi:10.1371/journal.ppat.1001310

There is growing interest in antiviral interferon-stimulated genes (ISGs) because of their potential as drug targets. In this paper, an overexpression screen has been used to assess the impact of several hundred ISGs on the replication of a number of viruses, including HIV-1 and hepatitis C virus. Combinations of validated antiviral ISGs were found to have additive effects and to converge on translational inhibition. Surprisingly, some ISGs actually enhance the replication of certain viruses, underlining the complexity of the response to interferon:
A diverse range of gene products are effectors of the type I interferon antiviral response. (2011) Nature 472, 7344. doi:10.1038/nature09907 (Subscription)
The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.

Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.

The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease. Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.

Viral Encephalomyelitis. 2011 PLoS Pathog 7(3): e1002004. doi:10.1371/journal.ppat.1002004
Encephalomyelitis resulting from virus infection of neurons is a disease that can be fatal or result in permanent disability due to irreversible damage of infected neurons. The immune response to infection can enhance neuronal damage or can control virus replication by noncytolytic mechanisms and thus determine outcome. However, noncytolytic virus clearance results in persistence of viral nucleic acid in the CNS and thus establishes a need for long-term local immune responses to prevent reactivation of infection and progressive disease. Understanding these mechanisms is necessary for development of strategies for treating and preventing neurologic disease due to viral encephalomyelitis.

Vaccines play a fundamental role in modern medicine and the introduction of Edward Jenner’s smallpox vaccine in 1798 marked an important turning point in the battle against infectious disease (Jenner, 1798). With the notable exceptions of smallpox and rabies, many of the early advances made in vaccinology during the 18th and 19th centuries were focused primarily on bacterial pathogens. These initial studies reflect the tools that were developed by early microbiologists to grow and study important pathogenic bacteria as well as some of the challenges faced by virologists prior to the advent of modern tissue culture technologies. During the 20th century, new viral vaccines against yellow fever, influenza, polio, measles, mumps, rubella, and others emerged. There are now 14 vaccines licensed in the USA that are directed against viral pathogens.

Contributions of humoral and cellular immunity to vaccine-induced protection in humans. Virology. (2011) 411(2): 206-215
Vaccines play a vital role in protecting the host against infectious disease. The most effective licensed vaccines elicit long-term antigen-specific antibody responses by plasma cells in addition to the development of persisting T cell and B cell memory. The relative contributions of these different immune cell subsets are context-dependent and vary depending on the attributes of the vaccine (i.e., live/attenuated, inactivated, and subunit) as well as the biology of the pathogen in question. For relatively simple vaccines against bacterial antigens (e.g., tetanus toxin) or invariant viruses, the immunological correlates of protection are well-characterized. For more complex vaccines against viruses, especially those that mutate or cause latent infections, it is more difficult to define the specific correlates of immunity. This often requires observational/natural history studies, clinical trials, or experimental evaluation in relevant animal models in order for immunological correlates to be determined or extrapolated. In this review, we will discuss the relative contributions of virus-specific T cell and B cell responses to vaccine-mediated protection against disease.

Related:

• How do you make a vaccine against Ebola virus?
• Dengue virus infection activates the unfolded protein response
• HIV vaccines – bad news, good news

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common respiratory pathogens. Both viruses comprise two genetic groups, A and B, distinguishable genetically and serologically which circulate with fluctuating frequencies. This gives rise to the observation of switching of the predominantly circulating subtype between seasons. Repeat HRSV infections occur throughout life with decreasing morbidity, and increasingly evidence suggests the same is also true for HMPV. In neither case has it yet been possible to make an effective vaccine against these troublesome pathogens.

Molecular Epidemiology and Evolution of Human Respiratory Syncytial Virus and Human Metapneumovirus. (2011) PLoS ONE 6(3): e17427
Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, the Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.

Related:

• Newly discovered respiratory viruses
• Human metapneumovirus glycoprotein G is an important virulence factor

Infectious diseases as a result of DNA virus infections are a major health concern worldwide. The major pathogenic DNA viruses include cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, polyoma virus and human papilloma virus. The two major species of herpesviruses such as CMV and HSV are clinically important. Herpes simplex virus is the cause of a wide range of diseases including some serious illnesses such as keratitis and encephalitis. Human cytomegalovirus is the major health risk in the newborn and in the immunocompromised causing congenital abnormalities and systemic diseases, respectively. Moreover, given the ability of DNA viruses to efficiently infect a wide range of cell types, these viruses also have gained clinical importance as potential gene delivery platforms to treat a variety of genetic diseases. The potent immune and inflammatory responses against the viral components however remain the stumbling block to the widespread clinical use of such vectors. Therefore a thorough mechanistic understanding of host anti-viral responses is central to the development not only of anti-viral therapeutics and vaccines but also in order to improve the safety of viral vectors in gene therapies.

Innate immune sensing of DNA viruses. Virology. Feb 17 2011
DNA viruses are a significant contributor to human morbidity and mortality. The immune system protects against viral infections through coordinated innate and adaptive immune responses. While the antigen-specific adaptive mechanisms have been extensively studied, the critical contributions of innate immunity to anti-viral defenses have only been revealed in the very recent past. Central to these anti-viral defenses is the recognition of viral pathogens by a diverse set of germ-line encoded receptors that survey nearly all cellular compartments for the presence of pathogens. In this review, we discuss the recent advances in the innate immune sensing of DNA viruses and focus on the recognition mechanisms involved.

Related:

• Toll-Like Receptors
• Virus tricks to grid-lock the type I interferon system

The recent discovery of miRNAs of viral origin has dramatically changed our view on virus-host interaction. Viral miRNAs have been shown to regulate genes of both cellular and viral origin, contributing to a favorable environment for the virus. However, the real importance of virus-encoded miRNAs during infection of their hosts remains elusive. This paper reports the first functional phenotype of a miRNA knock-out mutant of the mouse cytomegalovirus in vivo. It shows that the mutant virus is attenuated specifically in the salivary glands of infected mice, an organ essential for long-term persistence of the virus and host-to-host spread. This attenuation revealed a striking dependence on genetic background of the mice under study. Only combined depletion of natural killer and T cells abolished the phenotype. These results indicate that, by regulating the immune system, viral miRNAs may play an important role in an efficient persistent infection.

Cytomegalovirus microRNAs Facilitate Persistent Virus Infection in Salivary Glands. (2010) PLoS Pathog 6(10): e1001150. doi:10.1371/journal.ppat.1001150
Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets’ messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.

Related:

• Cytomegalovirus immune manipulation
• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Human cytomegalovirus and the cell cycle