Mosquito saliva enhances virus replication and disease

Biting mosquitoMosquito saliva, which is injected into the host as a mosquito probes for a blood vessel, contains a collection of chemicals which include anticoagulants to prevent blood clotting, vasodilators to keep blood vessels wide, and anesthetics to prevent us from sensing the mosquito. Saliva also contains components that enhance viral replication, dissemination, and pathogenesis by inducing an inflammatory response that inadvertently promotes infection by providing new cell targets for infection (paper link).

To separate the bite from virus inoculation, mice were first exposed to Aedes aegyptii mosquitoes, and then infected at the bite site with two different mosquito transmitted viruses, Semliki Forest virus or Bunyamwera virus. Mosquito bites caused more virus replication at the inoculation site, greater dissemination of virus, and more lethality compared with control mice that received only virus.

How does mosquito saliva enhance virus replication and dissemination? Part of the story is that as the mosquito probes for a blood vessel, it causes damage that leads to vascular leakage and accumulation of fluid (edema) which inhibits movement of virus to draining lymph nodes.

But delaying dissemination of virus alone does not promote infection and disease. Mosquito bites cause an infiltration of neutrophils (a type of white blood cell) into the bite site. The edema at the bite site is enhanced by neutrophils, because depleting these cells from mice greatly reduced edema. This depletion also returned viremia to levels observed in unbitten control mice, and restored dissemination of virus to draining lymph nodes. Neutrophils are not susceptible to infection with Semliki Forest virus, and therefore cannot explain the increase in virus replication at the bite site.

Enhanced virus replication in the skin occurs because the neutrophils elaborate chemokines that attract macrophages, which can be infected by Semliki Forest virus and Bunyamwera virus. One of the chemokines produced by neutrophils that is a macrophage attractant – CCL2 – binds a receptor on macrophages. Mice lacking the gene encoding the CCL2 receptor are protected from bite enhancement of Semliki Forest virus enhancement.

When a mosquito bites a host, it delivers saliva along with a virus. The saliva induces an inflammatory response and attracts neutrophils into the bite site. The resulting edema holds virus at the bite site until chemokines produced by neutrophils attract macrophages, which are then infected. The virus produced disseminates widely, reaching secondary tissues and causing disease.

It seems likely that the ability to replicate in macrophages that are recruited to the bite site is a property that was selected during evolution of mosquito-transmitted viruses. By replicating in macrophages, the amount of virus in the blood is increased, as well as the likelihood that the virus will be picked up by another mosquito and transmitted to a new host – a powerful selection mechanism. The down side – increased disease in the mammalian host – is an accidental side effect.

Think about that the next time you are scratching that raised bump on your skin caused by a mosquito bite.

Virus-induced fever might change bacteria from commensal to pathogen

Stem-loopNeisseria meningitidis may cause septicemia (bacteria in the blood) and meningitis (infection of the membrane surrounding the brain), but the bacterium colonizes the nasopharynx in 10-20% of the human population without causing disease. Although understanding how the bacterium changes from a commensal to a pathogen has been elusive, an important property is believed to be the ability to resist destruction by the immune response. Fever caused by a viral infection might be the trigger that makes N. meningitidis evade immunity.

A property of N. meningitidis that makes it cause disease is resistance to complement, a collection of proteins in the blood that help clear pathogens. N. meningitidis has evolved several mechanisms to avoid destruction by complement, including the production of a polysaccharide capsule, addition of sialic acid to a component of the bacterial outer membrane, and the production of a protein that binds one of the complement proteins. It is not clear why a commensal organism would have evolved such evasion mechansims – invading the blood and the brain are dead ends, as they do not lead to transmission to a new host.

An answer to this question comes from the finding that N. meningitidis proteins essential for resistance to complement are under the control of an RNA thermosensor. This control element is an RNA stem loop structure (pictured) formed by base pairing of local sequences within the RNA. Buried in the base-paired stem is a short sequence called the ribosome binding site that is essential for translation of the mRNAs into protein. At 30°C, the stem loop structure is intact, preventing binding of ribosomes to the mRNA; protein synthesis is blocked. At elevated temperatures – 37 or 40°C – abundant protein synthesis takes place, because the RNA secondary structure is denatured, allowing ribosomes to more efficiently access the ribosome binding site on the mRNA.

How do these findings explain why N. meningitidis becomes a pathogen? The temperature of the upper respiratory tract, where the bacterium normally colonizes, is low, so the RNA sensor is intact, preventing production of proteins needed for resistance to complement. If the respiratory tract is infected with a virus, a local immune response occurs which is accompanied by high temperatures – a fever. The RNA thermosensors of N. meningitidis have evolved to sense elevated temperature and turn on the synthesis of proteins that help it to avoid destruction by the immune response. Unfortunately, inflammation also damages the mucosal barriers that normally prevent microbes from invading the underlying tissues, where they have access to the bloodstream. N. meningitidis enters the blood stream, and because it is resistant to complement, it is not cleared. The result may be septicemia and infection of the brain. Moving away from its niche in the respiratory tract is probably not part of the microbe’s plan, but rather a consequence of the fact that it has evolved to survive immune responses to other pathogens in the respiratory tract.

There is some epidemiological support for this scenario: peaks of Neisseria meningitidis disease may follow outbreaks of influenza.

The conversion of commensal bacteria into pathogens by a second infection may be more common than we know. Streptococcus pneumoniae is a human nasopharyngeal commensal that colonizes 10 to 40% of healthy individuals. The bacterium is also a leading cause of respiratory disease. There is evidence that infection with influenza virus releases S. pneumoniae bacteria from biofilms; the free-living bacteria are then able to cause respiratory disease. One of the influenza virus-induced host signals responsible for changing S. pneumoniae from a commensal to a pathogen is fever.  For more on this story, listen to This Week in Microbiology #62.

Viruses of protozoan parasites may exacerbate human disease

TrichomonasMany protozoan parasites (Trichomonas, Leishmania, Giardia, Plasmodium, Entamoeba, Nagleria, Eimeria, Cryptosporidium) are infected with viruses. These viruses do not infect vertebrates, but their double-stranded RNA genomes are sensed by the innate immune system, leading to inflammatory complications of protozoan infections.

Trichomonas vaginalis is a protozoan parasite that infects the genitourinary tract of ~250 million individuals each year, leading to bacterial vaginosis, increased susceptibility to human immunodeficiency virus (HIV) and human papillomavirus (HPV), and reproductive complications such as infertility, pregnancy loss, and preterm delivery. Antibiotic treatment can clear the parasite but does not prevent some of these complications.

Half of T. vaginalis clinical isolates harbor dsRNA viruses (TVV) that do not harm their parasite host. It has been suggested that these TVVs are sensed by the human host, leading to inflammation and reproductive complications. To test this hypothesis, human epithelial cell cultures from the female reproductive tract were exposed to TVV-negative or positive T. vaginalis. Virus-positive parasites induced the production of interferon and proinflammatory cytokines while virus-negative parasites did not. The production of these cytokines is dependent upon Toll-like receptor 3 (TLR3), which is present on the inner endosome membrane. This observation suggests that virions or viral RNA released from T. vaginalis are taken into the cell by endocytosis where they encounter TLR3. In support of this idea, the addition of purified TVV virions to cells lead to TLR3-dependent production of cytokines.

To provide a plausible explanation for the failure of antibiotics to prevent T. vaginalis associated complications, protozoans with or without TVV were treated with metronidazole for 24 h, and the cell supernatant was added to cell cultures. Supernatants from the virus-containing, drug treated protozoa induced cytokines in a TLR3-dependent manner. Cytokines were not induced using supernatants of T. vaginalis lacking TVV, or from cells that were not treated with antibiotics. These results suggest that antibiotic treatment of virus-infected T. vaginalis causes an inflammatory response by liberating TVV virions or viral dsRNA from the protozoan.

These intriguing results show that protozoan viruses can be sensed by human cells, leading to an inflammatory response. In the vaginal mucosa this response is a double-edged sword: it can help limit infection but may also lead to pathological damage and increased susceptibility to bacterial and viral infection. Clinical studies are needed to determine if inflammation caused by TVV is linked to T. vaginalis associated disease and complications. The outcome of this work may determine whether the use of antivirals to inhibit TVV replication, or anti-inflammatory agents, should accompany antibiotic therapy to prevent the complications associated with T. vaginalis infection.

The complement system

The complement system is a collection of blood and cell surface proteins that is a major primary defense and a clearance component of innate and adaptive immune responses. At least 30 different complement proteins act sequentially to produce a wide ranges of activities, from cell lysis to augmentation of the adaptive response. The complement system has four major antimicrobial functions.

Lysis – Polymerization of specific activated complement components on a foreign cell or enveloped virus leads to the formation of pores. The lipid bilayer of the cell or virus is disrupted.

Activation of inflammation – Several peptides produced by proteolytic cleavage of complement proteins bind to vascular endothelial cells and lymphocytes. These cells then produce cytokines which stimulate inflammation and enhances responses to foreign antigens.

Opsonization – Certain complement proteins can bind to virions. Phagocytic cells with receptors for these complement proteins can then engulf the virus particles and destroy them. This process is called opsonization.

Solubilization of immune complexes – Some virus infections that are not cytopathic – the virus does not kill cells – lead to the accumulation of antibody-virus complexes. When these immune complexes lodge in blood vessels they can cause damage. An example is glomerulonephritis caused by deposition of antibody-antigen complexes in the kidney. Some complement proteins can disrupt these complexes and facilitate their clearance from the circulatory system.

There are three different complement pathways: classical, alternative, and mannan-binding. Unfortunately the nomenclature of the complement proteins is confusing, because they were named as they were discovered, not according to their function (see illustration below of the classical pathway). We’ll discuss the different pathways in the course of several posts. Don’t be daunted by the apparent complexity; stay with me and you’ll have a good appreciation of an extremely important part of our immune defense system.

And yes, viruses have evolved to modify the complement system.

classical-complement-s(click for a large version)

The inflammatory response

neutrophil-migrationDuring the earliest stages of a virus infection, cytokines are produced when innate immune defenses are activated. The rapid release of cytokines at the site of infection initiates new responses with far-reaching consequences that include inflammation.

One of the earliest cytokines produced is tumor necrosis factor alpha (TNF-α), which is synthesized by activated monocytes and macrophages. This cytokine changes nearby capillaries so that circulating white blood cells can be easily brought to the site of infection. TNF-α can also bind to receptors on infected cells and induce an antiviral response. Within seconds, a series of signals is initiated that leads to cell death, an attempt to prevent the spread of infection.

Inflammation is a very prominent response to TNF-α. There are four typical signs of inflammation: erythema (redness), heat, swelling, and pain. These are a consequence of increased blood flow and capillary permeability, the influx of phagocytic cells, and tissue damage. Increased blood flow is caused by constriction of the capillaries that carry blood away from the infected area, and leads to engorgement of the capillary network. Erythema and an increase in tissue temperature accompany capillary constriction. In addition, the permeability of capillaries increases, allowing cells and fluid to leave and enter the surrounding tissue. These fluids have a higher protein content than the fluids normally found in tissues, causing swelling.

Another feature of inflammation is the presence of immune cells, largely mononuclear phagocytes, which are attracted to the infected area by cytokines. Neutrophils are one of the earliest types of phagocytic cells that enter a site of infection, and are classic markers of the inflammatory response (illustrated). These cells are abundant in the blood, and usually absent from tissues. Together with infected cells, dendritic cells, and macrophages, they produce cytokines that can further shape the response to infection, and also modulate the adaptive response that may follow.

The precise nature of the inflammatory response depends upon the virus and the tissue that is infected. Viruses that do not kill cells – noncytopathic viruses – do not induce a strong inflammatory response. Because the cells and proteins of the inflammatory response come from the bloodstream, tissues with reduced access to the blood do not undergo the destruction associated with inflammation. However, the outcome of infection in such ‘privileged’ sites – the brain, for example – may be very different compared with other tissues.

As expected, the inflammatory response is highly regulated. One of the critical components is the ‘inflammasome’ – very large cytoplasmic structure with properties of pattern receptors and initiators of signaling (e.g. MDA-5 and RIG-I). Recent experimental findings demonstrate that the inflammasome is critical in innate immune response to influenza virus infection, and in moderating lung pathology in influenza pneumonia.

Thomas, P., Dash, P., Aldridge Jr., J., Ellebedy, A., Reynolds, C., Funk, A., Martin, W., Lamkanfi, M., Webby, R., & Boyd, K. (2009). The Intracellular Sensor NLRP3 Mediates Key Innate and Healing Responses to Influenza A Virus via the Regulation of Caspase-1 Immunity, 30 (4), 566-575 DOI: 10.1016/j.immuni.2009.02.006

Allen, I., Scull, M., Moore, C., Holl, E., McElvania-TeKippe, E., Taxman, D., Guthrie, E., Pickles, R., & Ting, J. (2009). The NLRP3 Inflammasome Mediates In Vivo Innate Immunity to Influenza A Virus through Recognition of Viral RNA Immunity, 30 (4), 556-565 DOI: 10.1016/j.immuni.2009.02.005