Around 30 years ago, researchers in the UK discovered DNA strands of herpes simplex virus 1 in postmortem brain samples of Alzheimer patients at much higher levels than in healthy brains, hinting that viral infection could be somehow involved in the disease.

Since then, a string of studies has bolstered the association between Alzheimer disease and HSV1, as well as other pathogens, particularly the herpesviruses HHV6A and HHV6B, yet proving causality has remained elusive.

Now, in an extensive screen of hundreds of diseased brains from three separate cohorts, a collaboration of US-based researchers reports no evidence for increased RNA or DNA levels of HHV6A or HHV6B in tissue from people with Alzheimer disease relative to that from healthy individuals, contradicting the results of some previous results. The scientists also failed to find an association between transcripts of other viruses that have been linked to Alzheimer, such as Epstein-Barr virus and cytomegalovirus, and Alzheimer, they report January 23 in Neuron.

“I’m very surprised,” Ruth Itzhaki, an Alzheimer disease researcher currently at the University of Oxford who was among those who first associated HSV1, and later HHV6, with the disease, writes to The Scientist in an email. “If their findings are correct, absence of HHV6 would make any involvement in Alzheimer disease unlikely,” although not impossible, she notes.

Several groups have reported the presence of HHV6 viruses in the brains of Alzheimer patients, most notably in a 2018 Neuron study. In that investigation, researchers had found higher levels of HHV6A in patients than in healthy controls, largely based on RNA and DNA sequencing data.

The result piqued the interest of Steven Jacobson, the chief of the viral immunology section at the National Institute of Neurological Disorders and Stroke, who says he was puzzled by certain aspects of the other team’s methodology. “I did not understand how they got these really interesting observations in that paper,” he tells The Scientist. Jacobson and his colleagues decided to investigate for themselves whether HHV6A or HHV6B were associated with Alzheimer disease.

Jacobson’s team first used RNA sequencing data to search for viral transcripts. For this, they made use of data that had been obtained from two different repositories: one consisting of 301 postmortem brain samples from the Mount Sinai Brain Bank, the second of 600 brain samples from the Religious Orders Study/Memory and Aging project. Both collections contained brains from both Alzheimer disease patients and healthy controls.

[The results] do increase my skepticism of herpes viruses in the brain being a causative agent in the disease.

—Tara Spires-Jones, Edinburgh University

The researchers then used an algorithm known as Pathseq, which is designed to plough through large amounts of human sequencing data and pick out microbial sequences, including 118 viruses. Interestingly, this analysis revealed no statistically significant difference in the amount of HHV6A or HHV6B RNA between diseased and healthy brains in either of the two cohorts. For instance, in the Religious Orders group, HHV6A was detected in only one of 173 brains with confirmed Alzheimer disease and one of the 158 age-matched controls. Screens for other viruses, including Epstein-Barr virus and several additional herpesviruses, also showed no significant difference between diseased brains or controls.

Then, taking a different approach, the team tested DNA samples that had been extracted from 708 healthy or diseased brains. (Around half came from the Religious Orders Study/Memory and Aging project; the other from the Johns Hopkins Brain Resource Center). Using a PCR technique, they searched for HHV6A or HHV6B viral DNA. Again, they found no significant difference between the Alzheimer disease samples and the controls.

Taken together, the results don’t support an association between HHV6 viruses and Alzheimer disease, but they also don’t rule it out, Jacobson explains. The absence or low levels of viral material at the time of death doesn’t mean that the viruses don’t play a role during early stages of the disease.

For instance, they could feasibly initiate the condition, and then stop proliferating and become latent as neurodegeneration progresses—a “hit and run” type of situation, notes Itzhaki, who wasn’t involved in the research. Given that latent HHV6 produces little RNA, “the chance of detecting latent [RNA] transcripts might be very low indeed,” she adds.

Supporting this notion, some recent work suggests that amyloid-β protein—which forms some of the tangles characteristic in Alzheimer’s disease—can form plaques around viruses such as HHV6, possibly as a way of protecting the brain against pathogens. “It’s possible” that this process could trigger the disease, Jacobson says.

Disparities in the literature

Ben Readhead, a data scientist at Arizona State University specializing in neurodegenerative diseases who wasn’t involved in the new research but has collaborations with two of its coauthors, says he is puzzled by its findings. He’s a coauthor of the 2018 study, which detected an abundance of several viral species, including HHV6, across the brain and linked them to different facets of Alzheimer’s disease pathology.

I think it is difficult to draw a strong conclusion from this study about whether HHV-6A/B is associated with Alzheimer’s disease.

—Ben Readhead, Arizona State University

Although that study was based on much of the same RNA sequencing data used by Jacobson’s group, the two teams report disparate results. Whereas in the RNA data from the Mount Sinai Brain Bank, Readhead’s team found HHV6A frequencies ranging from 18% to 35% of samples and HHV6B frequencies between 15% and 31%, Jacobson’s team found HHV6A or HHV6B frequencies ranging from 0% to 2.2%. “Another surprising finding is the absence of reported detection of several other very common, neurotropic viruses within the analysis of the RNA-sequence data sets,” Readhead notes in an email. (In a December 2019 letter in Neuron, another group of researchers take issue with the statistical robustness of Readhead’s study).

One reason for the disparities could be the different algorithms the teams used to analyze the RNA sequencing data. Perhaps the Pathseq tool isn’t suited to detecting the very low concentrations of viruses in a latent state, Readhead suggests. “I think it is difficult to draw a strong conclusion from this study about whether HHV-6A/B is associated with Alzheimer disease...without a clearer understanding of how to reconcile the unusually low frequencies of detected HHV-6A/B with a diverse scientific literature that supports a much higher prevalence,” he writes.

On the other hand, Tara Spires-Jones of Edinburgh University, a neuroscientist specializing in neurodegeneration who wasn’t involved in either study isn’t surprised by the findings from Jacobson’s group. “I thought this was a very solid study... [The results] do increase my skepticism of herpes viruses in the brain being a causative agent in the disease,” she says.

She questions whether reports of high virus levels in Alzheimer disease brains could be explained by the disease triggering viral entry into the brain, rather than the pathogens causing the disease. For instance, “there could be virus in the brain because of a disruption of the blood brain barrier that’s caused by disease,” she explains.

For Jacobson, his findings aren’t the end of the story. “In fact, we’re looking at even larger cohorts...to extend this study,” he says. The implication of a virus to be causatively involved in Alzheimer is huge, he says. “Those of us who do clinical trials, we really want to go in with our strongest pieces of evidence before we ask patients to take drugs and to do these really long expensive type of studies.”