A female Aedes albopictus mosquito taking a blood meal [Image courtesy of CDC/ San Gabriel Valley Mosquito & Vector Control District (SGVMVCD), Pablo Cabrera]

Studies in mice by scientists at Washington University School of Medicine in St. Louis suggest that the gut microbiome may play a role in the severity of infection by chikungunya virus (CHIKV). The studies showed that mice with faulty gut microbiomes were less able to generate certain immune responses to control infection by the mosquito-borne virus. In contrast, either recolonizing the animals’ intestines with a single Clostridium species, or giving the animals a secondary bile acid produced by the bacterium, improved immune responses, lowered levels of the virus in the mice’s blood, and even reduced the chances that a mosquito that fed on blood from infected mice would acquire the virus.

The findings indicate that a healthy microbiome could help to reduce the likelihood that an individual infected with chikungunya virus will develop severe disease, and could possibly even lessen community spread by disrupting the transmission of virus from person to mosquito to another person. “In many viral diseases, only a subset of the people who get infected become symptomatic, and we don’t really understand why,” said Michael S. Diamond, MD, PhD, the Herbert S. Gasser professor of medicine. “There might be things that happen during your lifetime that shape your immune system and influence whether you can stop the infection early and have minimal symptoms, or fail to stop it and develop severe disease. We found that when mice don’t have a healthy gut microbiome, not only do they get sicker, but mosquitoes that sample their blood are more likely to get infected. Promoting a healthy microbiome could be important not just for individuals who might get infected but for the whole community in breaking or reducing the cycle of transmission.”

Diamond is senior author of the team’s published paper in Cell, which is titled, “The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type 1 IFN Signaling Axis.”

Chikungunya virus is an alphavirus that is transmitted by mosquitoes. Historically confined to the Eastern Hemisphere, the virus has infected millions of people in the Americas since 2013. About half of all those infected with chikungunya virus never show symptoms, while others will develop fever and joint pain that lasts about a week, but 10–30% develop debilitating arthritis that persists for months or years. Scientists have understood little about why the severity of the illness varies so widely. “Although alphavirus viremia is a key determinant of disease severity and vector transmission, the factors modulating disease outcome remain poorly understood,” the authors wrote. “… little is known about the acquired and genetic factors that impact levels of virus in the human circulation.”

Gut bacteria metabolize and chemically modify the dietary compounds that pass through the digestive tract, generating vitamins and other compounds as byproducts, which are absorbed by the gut cells or taken up by other microbes, and help to regulate inflammation and the body’s response to infection. Growing evidence suggests that the intestinal microbiome can impact on antiviral immune responses not just in the gut, but at other sites in the body. The authors cite previous studies, which have shown that germ-free and antibiotic-treated mice exhibit “defects” in their innate and adaptive immune responses following infections with viruses including influenza and hepatitis B virus.

To find out if the gut microbiome affects the severity of chikungunya infection, Diamond, first author Emma Winkler, a graduate student in Diamond’s lab, and colleagues, studied germ-free mice, which had been kept under sterile conditions since birth and therefore never developed a gut microbiome, and ordinary laboratory mice treated with a cocktail of two commonly used antibiotics, to reduce the complexity of their gut microbiomes.

The researchers infected groups of these germ-free, and antibiotic-treated mice, with chikungunya virus. Groups of mice that harbored a normal microbiome were also infected with the virus, as controls. The researchers saw that the virus multiplied and spread rapidly in the mice that lacked a normal gut microbiome, reaching high levels in the blood and in tissues far from the site of infection. Further experiments showed that key immune cells were impaired in the mice with the faulty microbiomes, resulting in reduced type 1 interferon (IFN) responses. “Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN),” the investigators stated. “Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection.”

Introducing just one bacterial species—a normal member of the human gut microbiome known as Clostridium scindens—was enough to restore the ability of antibiotic-treated mice to fight the infection. “Remarkably, colonization of AV [ampicillin and vancomycin]-treated mice with C. scindens reduced CHIKV infection in serum and peripheral blood leukocytes,” the scientists stated. “… C. scindens colonization in AV-treated mice restored type I IFN levels in serum following CHIKV infection to those detected in vehicle-treated controls.”

C. scindens is not a bacterial species typically found in mice, but it is common in people, where it modifies a bile acid (BA) produced in the liver to generate a secondary bile acid that impacts on immune responses. Additional studies showed that giving just the modified bile acid to mice that lacked normal microbiomes was enough to restore type 1 interferon responses, and reduced viral levels in the blood and tissues. “These results demonstrate a functional role for microbiome-derived secondary BA in promoting MyD88-dependent type I IFN responses that limit CHIKV infection in circulation even in the context of an unperturbed microbiome,” the team noted.

“If having an unhealthy microbiome affects the virus levels in your blood, that raises an interesting question for a blood-borne pathogen,” continued Diamond, who is also a professor of molecular biology and of pathology and immunology. “Does the health of your microbiome impact transmission? It stands to reason that if there is more virus in the blood, a mosquito would be more likely to get infected when it takes a blood meal.”

To test this idea, Diamond and Winkler infected three groups of mice with chikungunya virus. One group was treated with antibiotics to eliminate their gut bacteria, a second was treated with antibiotics and later given C. scindens to repopulate such bacteria in their intestines, and the third group didn’t receive antibiotics at all, so they retained a normal gut microbiome. The researchers drew blood one day after infection and offered the blood to mosquitoes to feed on.

They found that more than half of the mosquitoes that sampled the blood of the antibiotic-treated mice became infected, compared with less than a third of the mosquitoes that fed on blood from the mice with normal microbiomes or with only C. scindens. “Thus, in the setting of an altered microbiome, colonization of mice with C. scindens can limit CHIKV viremia in a mammalian host and prevent transmission back to the mosquito vector,” the authors noted.

They say the study establishes a previously unknown role for the gut microbiome in promoting antiviral responses to alphavirus infection. “The finding that a specific bacterium and its associated metabolites can restrict alphavirus dissemination may provide insight into the variability of clinical disease in humans and enable approaches for mitigating alphavirus infection outcome and possibly, its transmission to mosquito vectors and epidemic spread.”

“There are plenty of people walking around with unhealthy microbiomes and varying levels of conjugated bile acids in their guts,” said Diamond. “There may be other bacteria that might be even better than C. scindens at modifying bile acids that could be used to rebalance microbiomes. If a probiotic like that was created, it might be one way to not only minimize disease in individuals, but reduce community spread at the same time.”

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