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Uncovering a Hidden Zika Outbreak in Cuba

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Zika Virus in Cuba
Credit: Sharon Isern, steampunkphage.com.

When Brazilian health officials discovered four years ago that the mosquito-borne Zika virus could cause severe birth defects and other serious health problems, it prompted a major effort across the Americas to curb the infection by controlling mosquitoes and issuing travel advisories. By mid-2017, the hard work seemed to have paid off, and reports of new Zika infections had nearly stopped.

But it turns out Zika may be tougher to control than once thought. New research shows that a large, previously hidden outbreak of Zika virus disease occurred in Cuba, just when it looked like the worst of the epidemic was over. The finding suggests that the Zika virus can linger over long periods, and that mosquito control efforts alone may slow, but not necessarily stop, the march of this potentially devastating infectious disease.

When combating global epidemics, it’s critical to track the spread of dangerous viruses from one place to the next. But some viruses can be tougher to monitor than others, and that certainly has been the case with Zika in the Americas. Though the virus can harm unborn children, many people infected with Zika never feel lousy enough to go to the doctor. Those who do often have symptoms that overlap with other prevalent tropical diseases, such as dengue and chikungunya fever, making it hard to recognize Zika.

That’s why in Brazil, where Zika arrived in the Americas by early 2014, this unexpected viral intruder went undetected for well over a year. By then, it had spread unnoticed to Honduras, circulating rapidly to other Central American nations and Mexico—likely by late 2014 and into 2015.

In the United States, even with close monitoring, a small local outbreak of Zika virus in Florida also went undetected for about three months in 2016 [1]. Then, in 2017, Florida officials began noticing something strange: new cases of Zika infection in people who had traveled to Cuba.

This came as a real surprise because Cuba, unlike most other Caribbean islands, was thought to have avoided an outbreak. What’s more, by then the Zika epidemic in the Americas had slowed to a trickle, prompting the World Health Organization to delist it as a global public health emergency of international concern.

Given the Cuban observation, some wondered whether the Zika epidemic in the Americas was really over. Among them was an NIH-supported research team, including Nathan Grubaugh, Yale School of Public Health, New Haven, CT; Sharon Isern and Scott Michael, Florida Gulf Coast University, Fort Myers; and Kristian Andersen, The Scripps Research Institute, La Jolla, CA, who worked closely with the Florida Department of Health, including Andrea Morrison.

As published in Cell, the team was able to document a previously unreported outbreak in Cuba after the epidemic had seemingly ended [2]. Interestingly, another research group in Spain also recently made a similar observation about Zika in Cuba [3].

In the Cell paper, the researchers show that between June 2017 and October 2018, all but two of 155 cases—a whopping 98 percent of travel-associated Zika infections—traced back to Cuba. Further analysis suggests that the outbreak in Cuba was likely of similar magnitude to outbreaks that occurred in other Caribbean nations.

Their estimates suggest there were likely many thousands of Zika cases in Cuba, and more than 5,000 likely should have been diagnosed and reported in 2017. The only difference was the timing. The Cuban outbreak of Zika virus occurred about a year after infections subsided elsewhere in the Caribbean.

To fill in more of the blanks, the researchers relied on Zika virus genomes from nine infected Florida travelers who returned from Cuba in 2017 and 2018. The sequencing data support multiple introductions of Zika virus to Cuba from other Caribbean islands in the summer of 2016.

The outbreak peaked about a year after the virus made its way to Cuba, similar to what happened in other places. But the Cuban outbreak was likely delayed by a year thanks to an effective mosquito control campaign by local authorities, following detection of the Brazilian outbreak. While information is lacking, including whether Zika infections had caused birth defects, it’s likely those efforts were relaxed once the emergency appeared to be over elsewhere in the Caribbean, and the virus took hold.

The findings serve as yet another reminder that the Zika virus—first identified in the Zika Forest in Uganda in 1947 and for many years considered a mostly inconsequential virus [4]—has by no means been eliminated. Indeed, such unrecognized and delayed outbreaks of Zika raise the risk of travelers innocently spreading the virus to other parts of the world.

The encouraging news is that, with travel surveillance data and genomic tools —enabled by open science—it is now possible to detect such outbreaks. By combining resources and data, it will be possible to develop even more effective and responsive surveillance frameworks to pick up on emerging health threats in the future.

In the meantime, work continues to develop a vaccine for the Zika virus, with more than a dozen clinical trials underway that pursue a variety of vaccination strategies. With the Zika pandemic resolved in the Americas, these studies can be harder to conduct, since proof of efficacy is not possible without active infections. But, as this paper shows, we must remain ready for future outbreaks of this unique and formidable virus.

References:

[1] Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Grubaugh et al. Nature. 2017 Jun 15;546(7658):401-405.

[2] Travel surveillance and genomics uncover a hidden Zika outbreak during the waning epidemic. Grubaugh ND, Saraf S, Gangavarapu K, Watts A, Tan AL, Oidtman RJ, Magnani DM, Watkins DI, Palacios G, Hamer DH; GeoSentinel Surveillance Network, Gardner LM, Perkins TA, Baele G, Khan K, Morrison A, Isern S, Michael SF, Andersen .KG, et. al. Cell. 2019 Aug 22;178(5):1057-1071.e11.

[3] Mirroring the Zika epidemics in Cuba: The view from a European imported diseases clinic. Almuedo-Riera A, Rodriguez-Valero N, Camprubí D, Losada Galván I, Zamora-Martinez C, Pousibet-Puerto J, Subirà C, Martinez MJ, Pinazo MJ, Muñoz J. Travel Med Infect Dis. 2019 Jul – Aug;30:125-127.

[4] Pandemic Zika: A Formidable Challenge to Medicine and Public Health. Morens DM, Fauci AS. J Infect Dis. 2017 Dec 16;216(suppl_10):S857-S859.

Links:

Video: Uncovering Hidden Zika Outbreaks (Florida Gulf Coast University, Fort Myers)

Zika Virus (National Institute of Allergy and Infectious Diseases/NIH)

Zika Virus Vaccines (NIAID)

Zika Free Florida (Florida Department of Health, Tallahassee)

Grubaugh Lab (Yale School of Public Health, New Haven, CT)

Andersen Lab (The Scripps Research Institute, La Jolla, CA)

NIH Support: National Institute of Allergy and Infectious Diseases; National Center for Advancing Translational Sciences


Enlisting CRISPR in the Quest for an HIV Cure

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Today, thanks to remarkable advances in antiretroviral drugs, most people with the human immunodeficiency virus (HIV) can expect to live an almost normal lifespan. But that means staying on medications for life. If those are stopped, HIV comes roaring back in just weeks. Finding a permanent cure for HIV infection, where the virus is completely and permanently eliminated from the body, has proven much tougher. So, I’m encouraged by recent work that shows it may be possible to eliminate HIV in a mouse model, and perhaps—with continued progress—someday we will actually cure HIV in humans.

This innovative approach relies on a one-two punch: drugs and genetic editing. First, HIV-infected mice received an experimental, long-acting form of antiretroviral therapy (ART) that suppresses viral replication. This step cleared the active HIV infection. But more was needed because HIV can “hide” by inserting its DNA into its host’s chromosomes—lying dormant until conditions are right for viral replication. To get at this infectious reservoir, researchers infused the mice with a gene-editing system designed to snip out any HIV DNA still lurking in the genomes of their spleen, bone marrow, lymph nodes, and other cells. The result? Researchers detected no signs of HIV in more than one-third of mice that received the combination treatment.

The new study in Nature Communications is the product of a collaboration between the NIH-funded labs of Howard Gendelman, University of Nebraska Medical Center, Omaha, and Kamel Khalili, Temple University, Philadelphia [1]. A virologist by training, Khalili years ago realized that HIV’s ability to integrate into the genomes of its host’s cells meant that the disease couldn’t be thought of only as a typical viral infection. It had a genetic component too, suggesting that an HIV cure might require a genetic answer.

At the time, however, the tools to remove HIV DNA from human cells without harming the human genome weren’t available. That’s changed in recent years with the discovery and subsequent development of a very precise gene-editing tool known as CRISPR/Cas9.

CRISPR/Cas9 editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome, where it can be used to cut out, replace, or repair disease-causing mutations. Efforts are underway to apply CRISPR/Cas9 to the treatment of sickle cell disease, muscular dystrophy, and more.

Could CRISPR/Cas9 also remove HIV DNA from infected cells and eliminate the infection for good? Such an approach might be particularly helpful for people on ART who have persistent HIV DNA in the cells of their cerebrospinal fluid. A recent NIH-funded study in Journal of Clinical Investigation found that an association between this HIV reservoir and neurocognitive difficulties [2]

Earlier work by Khalili’s team showed that CRISPR could indeed remove HIV DNA from the genomes of host cells [3]. The problem was that, when delivered on its own, CRISPR couldn’t snip out every last bit of viral DNA from all cells as needed to get rid of HIV completely and permanently. It was crucial to reduce the burden of HIV genomes to the lowest possible level.

Meanwhile, Gendelman’s lab had been working to develop a new and more effective way to deliver ART. Often delivered in combinations, standard ART drugs are effective in suppressing HIV replication. However, people need to take their oral medications daily without fail. Also, most ART triple therapy drugs are water soluble, which means its cocktail of medications are swiftly processed and excreted by the body without reaching many places in the body where HIV hides.

In his quest to make ART work more effectively with fewer doses, Gendelman’s team altered the chemical composition of antiretroviral medicines, generating fat-soluble drug nanocrystals. The nanocrystals were then packaged into nanoparticles and delivered by intramuscular injection. The new drug formulation, known as long-acting slow-effective release (LASER) ART, reaches lymph nodes, spleen, gut, and brain tissues where HIV lurks [4]. Once there, it’s stored and released slowly over time. Still, like conventional ART, LASER ART can never completely cure HIV.

So, Gendelman teamed up with Khalili to ask: What would happen if LASER ART was followed by a round of CRISPR/Cas9? In a series of studies, the researchers tested LASER ART and CRISPR/Cas9, both alone and in combination. A total of 23 HIV-infected mice engineered to have some “humanized” immune features received the experimental combination therapy.

As expected, neither LASER ART nor CRISPR/Cas9 by itself proved sufficient to eradicate HIV in the mice. However, when LASER ART and CRISPR/Cas9 were delivered sequentially, the results were much different. Researchers found no evidence of HIV in the spleens or other tissues of more than one-third of the sequentially treated animals.

It’s important to note that this gene-editing approach to eradicating HIV is being applied to non-reproductive cells (somatic). The NIH does not support the use of gene-editing technologies in human embryos (germline) [5].

Of course, mice, even with humanized immune systems, are not humans. More research is needed to replicate these findings and to figure out how to make this approach to HIV treatment more effective in animal models before we can consider moving into human clinical trials. Still, these findings do provide a new reason for increased hope that an actual cure may ultimately be found for the tens of millions of people in the United States and around the globe now living with HIV.

References:

[1] Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice. Dash PK, Kaminski R, Bella R, Su H, Mathews S, Ahooyi TM, Chen C, Mancuso P, Sariyer R, Ferrante P, Donadoni M, Robinson JA, Sillman B, Lin Z, Hilaire JR, Banoub M, Elango M, Gautam N, Mosley RL, Poluektova LY, McMillan J, Bade AN, Gorantla S, Sariyer IK, Burdo TH, Young WB, Amini S, Gordon J, Jacobson JM, Edagwa B, Khalili K, Gendelman HE. Nat Commun. 2019 Jul 2;10(1):2753.

[2] Spudich S et al. Persistent HIV-infected Cells in Cerebrospinal Fluid are Associated with Poorer Neurocognitive Performance. J Clin Invest. 2019. DOI: 10.1172/JCI127413 (2019).

[3] In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Yin C, Zhang T, Qu X, Zhang Y, Putatunda R, Xiao X, Li F, Xiao W, Zhao H, Dai S, Qin X, Mo X, Young WB, Khalili K, Hu W. Mol Ther. 2017 May 3;25(5):1168-1186.

[4] Creation of a nanoformulated cabotegravir prodrug with improved antiretroviral profiles. Zhou T, Su H, Dash P, Lin Z, Dyavar Shetty BL, Kocher T, Szlachetka A, Lamberty B, Fox HS, Poluektova L, Gorantla S, McMillan J, Gautam N, Mosley RL, Alnouti Y, Edagwa B, Gendelman HE. Biomaterials. 2018 Jan;151:53-65.

[5] Statement on Claim of First Gene-Edited Babies by Chinese Researcher. The NIH Director, NIH. 2018 November 28.

Links:

HIV/AIDS (National Institute of Allergy and Infectious Diseases/NIH)

HIV Treatment: The Basics (U.S. Department of Health and Human Services)

Fast Facts (HIV.gov)

Global Statistics (HIV.gov)

Kamel Khalili (Temple University, Philadelphia, PA)

Howard Gendelman (University of Nebraska Medical Center, Omaha)

NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute of Allergy and Infectious Diseases; National Institute on Aging; National Institute on Drug Abuse; Common Fund


Looking to Llamas for New Ways to Fight the Flu

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Lllama nanobodiesResearchers are making tremendous strides toward developing better ways to reduce our risk of getting the flu. And one of the latest ideas for foiling the flu—a “gene mist” that could be sprayed into the nose—comes from a most surprising source: llamas.

Like humans and many other creatures, these fuzzy South American relatives of the camel produce immune molecules, called antibodies, in their blood when exposed to viruses and other foreign substances. Researchers speculated that because the llama’s antibodies are so much smaller than human antibodies, they might be easier to use therapeutically in fending off a wide range of flu viruses. This idea is now being leveraged to design a new type of gene therapy that may someday provide humans with broader protection against the flu [1].


A Lean, Mean DNA Packaging Machine

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Three views of bacteriophage T4

Credit: Victor Padilla-Sanchez, The Catholic University of America, Washington, D.C.

All plants and animals are susceptible to viral infections. But did you know that’s also true for bacteria? They get nailed by viruses called bacteriophages, and there are thousands of them in nature including this one that resembles a lunar lander: bacteriophage T4 (left panel). It’s a popular model organism that researchers have studied for nearly a century, helping them over the years to learn more about biochemistry, genetics, and molecular biology [1].

The bacteriophage T4 infects the bacterium Escherichia coli, which normally inhabits the gastrointestinal tract of humans. T4’s invasion starts by touching down on the bacterial cell wall and injecting viral DNA through its tube-like tail (purple) into the cell. A DNA “packaging machine” (middle and right panels) between the bacteriophage’s “head” and “tail” (green, yellow, blue spikes) keeps the double-stranded DNA (middle panel, red) at the ready. All the vivid colors you see in the images help to distinguish between the various proteins or protein subunits that make up the intricate structure of the bacteriophage and its DNA packaging machine.


Twinkle, Twinkle Little Cryo-EM Star

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The stars are out and shining this holiday season. But there are some star-shaped structures now under study in the lab that also give us plenty of reason for hope. One of them is a tiny virus called bacteriophage phi-6, which researchers are studying in an effort to combat a similar, but more-complex, group of viruses that can cause life-threatening dehydration in young children.

Thanks to a breakthrough technology called cryo-electron microscopy (cryo-EM), NIH researchers recently captured, at near atomic-level of detail, the 3D structure of this immature bacteriophage phi-6 particle in the process of replication. At the points of its “star,” key proteins (red) are positioned to transport clipped, single-stranded segments of the virus’ own genetic information into its newly made shell, or procapsid (blue). Once inside the procapsid, an enzyme (purple) will copy the segments to make the genetic information double-stranded, while another protein (yellow) will help package them. As the procapsid matures, it undergoes dramatic structural changes.


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