Mosquito 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.

The TWiV team is together in New York City for a conversation with Nobel Laureate Harold Varmus about his remarkable career in science.

You can find TWiV #400 at microbe.tv/twiv, or listen/watch below.

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Flaviviruses are unusual because antibodies that cross-react with different viruses can enhance infection and disease. This property, called antibody-dependent enhancement or ADE, has been documented to occur among the four serotypes of dengue virus. It has implications for infection with or vaccination against Zika virus or dengue virus.

Earlier this year (virology blog link) it was shown that antibodies to dengue virus – in the form of serum from infected patients, or two human monoclonal antibodies – bind to Zika virus and can enhance infection of Fc-receptor bearing cells (Fc receptors bind the antibody molecule, allowing uptake into cells – illustrated). When the antibodies to dengue virus were omitted, Zika virus barely infected these cells. The conclusion is that dengue antibodies enhance infection of cells in culture by Zika virus.

This early work was first published as a preprint on the bioRxiv server – which lead some to criticize me for discussing the work before peer review. However, I subjected the paper to my own peer review, of which I am entirely capable, and decided it was worthy of discussion on this blog.

The results have now been confirmed by an independent group (paper link). Sera from patients that were infected with dengue virus, as well as dengue virus specific human monoclonal antibodies, were shown to bind Zika virus and enhance infection of Fc receptor bearing cells. These are the same findings of the group who first published on bioRxiv. That paper still has not been published – apparently it is mired in peer review, with many new experiments requested. I do hope that none of the authors of the second paper are involved in delaying its publication – something that happens all too often in science. As a colleague once remarked, ‘the main function of peer review is to prevent your competitors from publishing their work’.

Whether or not antibodies to dengue virus enhance Zika virus disease in humans is an important unanswered question.

If you are wondering whether antibodies to Zika virus can enhance dengue virus infection, the answer is yes (paper link). Monoclonal antibodies were isolated from four Zika virus-infected patients, and shown to enhance infection of Fc receptor bearing cells with either Zika virus or dengue virus. Furthermore, administration of these antibodies to mice before infection with dengue virus led to severe disease and lethality, a demonstration of antibody-dependent enhancement in an animal model.

Of interest is the finding that ADE mediated lethality in this mouse model can be completely prevented by co-administering the same antibody that has been modified to block binding to Fc receptors on cells. This result suggests a modality for treating patients with enhanced disease caused by either dengue virus or Zika virus.

These observations suggest that we need to be careful when deploying vaccines against Zika virus or dengue virus – it is possible that the antibody response could enhance disease. Recently a dengue virus vaccine called Dengvaxxia was approved for use in Brazil, Mexico, and the Philippines. However, the vaccine is not licensed for use in children less than 9 years of age because in clinical trials, immunization lead to more severe disease after infection compared with non-immunized controls. Analysis of the clinical trial data (paper link) indicates that seronegative individuals of all ages were at increased risk for developing severe disease that requires hospitalization. The authors suggest that severe disease is a consequence of enhancement of infection caused by antibodies induced by the vaccine (see CIDRAP article for more information).

These observations lead to the question of whether immunization against dengue and Zika viruses might enhance disease caused by either virus. Could a solution to this potential problem be to use a vaccine that combines the four serotypes of dengue virus with Zika virus? If so, the dengue virus component should not be Dengvaxia, but possibly another vaccine (e.g. TV003 – virology blog link) that does not induce disease enhancing antibodies.

The first experimental Zika virus vaccine has been published, and in this episode of Virus Watch, I explain how it works – it’s a DNA vaccine – and I compare it with all the other vaccines out there.

The latest Zika virus news from the ConTWiVstadors, including a case of female to male transmission, risk of infection at the 2016 summer Olympics, a DNA vaccine, antibody-dependent enhancement by dengue antibodies, and sites of replication in the placenta.

You can find TWiV #399 at microbe.tv/twiv, or listen below.

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Viruses that replicate in the male or female reproductive tract are considered to be potential causes of human infertility. Several herpesviruses have been implicated in male infertility, and now human herpesvirus 6A (HHV-6A) has been found in endometrial cells of women with unexplained infertility (paper link).

HHV-6 was only recently discovered (1986) and is now known to occur as two variants, HHV-6A and HHV-6B. The latter is a major cause of exanthem subitum, a rash of infants, but no disease has been clearly associated with HHV-6A. These viruses are transmitted to infants early in life via saliva, from mother to child, from siblings, or from other infants at day care centers. Seroprevalence studies indicate that almost all children are infected with these viruses by 2 years of age.

To determine if HHV-6 might be a cause of infertility, a study (paper link) was conducted of 30 women with unexplained primary fertility, and 36 women with at least one previous pregnancy. HHV-6B DNA was detected in PBMC from both infertile and fertile groups (25 and 28%, respectively); HHV-6A DNA was not detected. In contrast, endometrial epithelial cells from 13/30 (43%) infertile women were positive for HHV-6A DNA; this viral DNA was not detected in endometrium of fertile women. When placed in culture, endometrial epithelial cells produced viral early and late proteins, suggesting the presence of infectious virus.

Presence of HHV-6A DNA in endometrial epithelial cells was also associated with an altered hormonal and immune environment. Estradiol levels were higher in infected versus uninfected infertile women. The authors suggest that higher levels of this hormone could be involved in allowing HHV-6A infection of the endometrium.

Levels of a specific type of uterine NK cell were lower in HHV-6A positive women, and IL-10 (a Th2 cytokine) was elevated while IFN-gamma (a Th1 cytokine) was decreased. There were no differences in the levels of these cells and cytokines in peripheral blood. These changes are consistent with an increase in the ratio of Th1/Th2 responses that has been documented in female infertility.

The authors also observed enhanced endometrial NK cell responses to HHV-6A in infected but not uninfected women, together with an increase in the number of these cells that are activated when cultured with HHV-6A infected cells.

I wonder what was the source of HHV-6A in the endometrium, as the virus was not detected in blood. Was the infection recently acquired, or did it occur years before, with the virus establishing a chronic infection in the uterus?

The results suggest that HHV-6A infection of the endometrium triggers an abnormal NK cell and cytokine profile, which in turn leads to a uterine environment that is not compatible with fertility. The results need to be confirmed with studies of additional fertile and infertile women. It would also be useful to have an animal model of HHV-6A infection of the endometrium, which could lead to mechanistic work to determine how virus infection causes infertility.

Image: Electron micrograph of HHV-6 (image credit)

Vincent speaks with Sandy Weller about her career and her work on the mechanisms of synthesis, maturation, cleavage and packaging of viral DNA genomes.

You can find TWiV 398 at microbe.tv/twiv, or listen/watch below.

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Andrew W. Gurman, M.D., President of the American Medical Association, has expressed disappointment in the failure of Congress to support the US public health response to Zika Virus:

At a time when concerns continue to mount about the nation’s readiness to protect the public from the Zika virus, the AMA is disappointed by Congress’ failure to pass legislation before adjourning for summer recess that would provide the resources necessary for our country to respond to this looming public health crisis.

Without ensuring there are sufficient resources available for research, prevention, control and treatment of illnesses associated with the Zika virus, the United States will be ill-equipped to deploy the kind of public health response needed to keep our citizens safe and healthy—especially since the spread of mosquito-borne illness is accelerated during the summer months.

I could not agree more with the AMA – Congress has so far failed to do the right thing with respect to Zika virus. Even if the virus does not spread within the continental United States, this country has an obligation to be a leader in scientific research that would benefit the entire world. Zika virus is clearly a threat to other parts of the globe, and by refusing to fund research on this virus, Congress is sending the message that it doesn’t care about the health problems of others.

Vincent speaks with Peter Palese about his illustrious career in virology, from early work on neuraminidases to universal influenza virus vaccines, on episode #396 of the science show This Week in Virology.

You can find TWiV #396 at microbe.tv/twiv, or listen below.

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