Are some virus loads introduced to human cell but never triggered?

Are some virus loads introduced to human cell but never triggered?

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Is there a term or any evidence of phage DNA integrating into chromosomes/ DNA but never being triggered?

For example, could a virus that affected Neanderthals still infect human cells today but is never expressed, triggered, or executed?

How often is latent viral DNA ignored by the host?

There is viral DNA within our genomes. Embedded in the genomes of all vertebrates are the proviral remnants of previous retroviral infections. It is possible that some have conferred biological benefits.

Human endogenous retroviruses (HERVs) represent the proviral phase of exogenous retroviruses that have integrated into the germ line of their host. They are transmitted vertically through the germline and are thus inherited by successive generations in a Mendelian manner.

HERVs possess a similar genomic organisation to present day exogenous retroviruses such as human immunodeficiency virus (HIV) and human T cell leukaemia virus.

Although many are defective through the accumulation of mutations, deletions, and termination signals, some HERVs have been implicated in certain autoimmune diseases and cancers and might have a role in the etiology and pathology of disease.

Within humans, the most recently active ERVs are members of the HERV-K (HML2) family. This family first integrated into the genome of the common ancestor of humans and Old World monkeys at least 30 million years ago, and it contains >12 elements that have integrated since the divergence of humans and chimpanzees, as well as at least two that are polymorphic among humans.

Here we show that the HERV-K (HML2) family has increased in copy number predominantly via reinfection, and that the family has probably retained replication-competent and infectious members for >30 million years. We also present evidence for persistent reinfection by other ERV families within the human genome, and suggest that endogenous retrovirus families are often capable of extremely long periods of smoldering infection.

Long-term reinfection of the human genome by endogenous retroviruses
The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences
Demystified… Human endogenous retroviruses

Viral vectors travel longer distances than previously thought

Where viral vectors "travel" and which types of neural cells they infect, can be visualized by fluorescent Proteins being transmitted. Credit: Kirsti Witter/Vetmeduni Vienna

Gene transfer is seen as a hopeful therapy for Alzheimer's and Parkinson's patients. The approach involves using harmless laboratory-produced viruses to introduce important genes into the brain cells. In a study on mice, a team of researchers from Vetmeduni Vienna for the first time investigated how far these viruses spread in the brain and which cells they infect. Some of the artificial viruses travelled from the injection site in the brain as far as the olfactory bulb or the cerebellum and infected not only neurons but also other cells. The results, which were published in the journal Histochemistry and Cell Biology, could help to improve the selection of suitable viral "gene transporters" for custom therapies using gene transfer.

Purposefully infecting brain cells with viruses may seem somewhat odd. But for patients suffering from neurodegenerative diseases such as Alzheimer's or Parkinson's, this type of therapy could be a glimmer of hope. The viruses used in this approach do not trigger any disease themselves. They serve as harmless transporters for genes specifically intended to treat these disorders. The therapy, called gene transfer, uses the ability of viruses to insert their genes into the genome of a host cell. This method could therefore be used to purposefully introduce helpful genetic information into neurons.

Viral vectors don't stay put

Viruses suitable for gene transfer are injected into the brain. Previously, however, there had been no studies of how far the viral transporters can spread from the injection site. Earlier studies had usually only investigated the immediate area around the injection canal. A new study with mice has now shown for the first time that some of the tested viruses can travel long distances into different areas of the brain. "In our study, we injected the viral vectors into key areas of the cerebrum responsible, among other things, for the coordinationof body movement ," explains Kirsti Witter from the Institute for Anatomy, Histology and Embryology at Vetmeduni Vienna. From there, some of the viruses spread into distant areas such as the cerebellum or the olfactory bulb.

"This information is important because, depending on the type of neurodegenerative disease, it may be desirable to have as broad a distribution of the virus as possible or to infect a specific, strictly delimited area," says first author Juraj Hlavaty. "This study also shows that all tested viruses can infect the neurons and the surrounding glial cells as expected. Depending on the type of virus, however, there were differences in the number and ratio of the infected cell types."

Inflammation could influence which brain cells are infected

Depending on the virus strain used, the injection triggered a mild or more pronounced reaction of the nerve tissue in the treated mice. The stronger the immune response, the more glial cells were infected. "The fact that individual viruses infected these cells better than the neurons must, however, still be confirmed in future experiments," says Hlavaty.

The results of the work, achieved in collaboration with the University of West Bohemia, Pilsen, Czech Republic, and the Paul-Ehrlich-Institute, Langen, Germany, should contribute to improve the selection of viral transporters. "The goal is to create a toolbox of possible viruses in order to choose exactly the right transporter for the custom treatment of a neurodegenerative disease," says Witter.

Artificial copies of viruses as hopeful therapy

Copies of lentiviruses are especially well-suited for gene transfer therapy. "The genome of laboratory-produced lentiviruses consists only of areas that are necessary for the infection and incorporation into the genome. This represents a fundamental difference between these viruses and naturally occurring pathogenic viruses," explains Hlavaty. Through the ability of the artificial viruses to enter a host, the inserted human genes are introduced into the infected cells to assume the tasks that the patients' cells no longer perform themselves.


Hepatitis C Virus (HCV) is a major pathogen given its extremely high prevalence (around 350 millions of chronically infected individuals worldwide), the high rate of chronic infection and the significant risk of severe chronic active hepatitis and cirrhosis among chronically infected subjects. HCV infection induces chronic infection in up to 60–80% of infected adults. The pathogenic effects of chronic HCV infections is highly variable some patients will only show minimal liver lesions while others (around 20%) will develop after 5–10 years follow-up severe fibrosis and cirrhosis. Finally, 30–50% of patients with cirrhosis, whatever the etiological factor, develop HCC after a 10 year follow-up.

Acute and chronic hepatitis induced by most hepatitis viruses clearly implicate the host immune response to viral proteins. The development of molecular biology has led to realize that some hepatitis viruses also directly modulate liver cell proliferation and viability. This issue has important implications for understanding the molecular bases of the liver lesions and liver carcinogenesis, as well as for developing new therapeutical strategies. The present review will focus on the viral pathogenetic mechanisms based on the interaction of HCV proteins with host cellular signaling transduction pathways regulating cell growth and viability and on the strategies developed by the virus to persist in the host and escape to antiviral therapy.

Conceptualizing the interaction between the host immune response and the virus

Looking at the current pandemic entirely from a biological perspective and assuming that the majority of humankind does not have an extended cross-reaction to other viruses of the coronavirus family ( Braun etਊl., 2020 Grifoni etਊl., 2020 Mateus etਊl., 2020 ), the introduction of SARS-CoV-2 into the human population is one of the biggest evolutionary events in the last hundred years ( Morens and Fauci, 2020 Morens etਊl., 2020 ). In such an evolutionary experiment, the innate immune system must be assigned a very special role in the defense against SARS-CoV-2 ( Amor etਊl., 2020 Mantovani and Netea, 2020 Vabret etਊl., 2020 ). An important part of innate immunity is the cell-autonomous response of the cells that get infected by the virus, which is influenced by the biology of the receptors and co-receptors for viral entry ( Hoffmann etਊl., 2020 Wu etਊl., 2020 Yao etਊl., 2020 ), as well as all cellular mechanisms that determine the viral life cycle ( Cyranoski, 2020 ). In the SARS-CoV-2 pandemic, the virus encounters the human population as a swarm of genetic variants and the processes of infection and virus replication may still be subject to major genetic changes, by which certain virus variants may achieve an evolutionary advantage. These processes are in full swing and accelerate as more individuals become infected ( Callaway, 2020 Cyranoski, 2020 Korber etਊl., 2020 ). It therefore will be important to monitor infected individuals for potential changes in the very early steps of the immune response to the virus, mainly triggered by infected cells and early interactions with adjacent innate immune cells.

Conceptually, when addressing important defense mechanisms of the host, it is important to consider all parameters that may be relevant for such an initial interaction of a new virus with a species ( Figureਁ ). Bearing in mind that most of the steps of the interaction between the virus and the host will follow a normal distribution of attributes or parameters required to describe the outcome of the interaction, it is not entirely surprising that the clinically observed disease courses show an enormous heterogeneity ( Berlin etਊl., 2020 Gandhi etਊl., 2020 ). For example, the induction of an interferon response by an infected cell might follow a normal distribution in the population with low, intermediate, and high responders thereby triggering different magnitudes of cellular responses and thus very different downstream effects (e.g., in the innate immune system). In Figureਁ , we illustrate important innate immune mechanisms that might be particularly prone to heterogeneous outcomes𠅍ue to both environmental and genetic factors—within the human population and therefore these should be a major focus in our efforts to determine the role of the innate immune system for infectivity, viral spreading, and disease course but also long-term outcome. We postulate that such conceptualization of the interplay between the innate immune system and the virus will help to focus on the most critical steps first, which then can be validated in larger studies.

Conceptualizing the interaction between the host immune response and the virus

Proposed fields of research along the disease trajectory in five phases that influence pathophysiology with an emphasis on innate immunity. Methodologies suggested to be applied for addressing certain areas are represented as color-coded circles. These reflect frequently used methods in previously published studies on COVID-19. This is only a selection and we make no claim to completeness. The overall concept could be extended to the adaptive immune system and other organ systems. CyTOF, cytometry by time of flight, mass cytometry ELISA OLINK, plasma proteome by proximity extension assay scRNA-seq, single-cell RNA sequencing seq, sequencing WGS, whole genome sequencing.

As we will discuss, hypothesis-driven systems medicine approaches (Box 1) have a very high chance to quickly uncover the most critical and variable steps in the interactions between the virus and the host’s immune system to link them to the different clinical phenotypes allowing a better stratification of patients that will foster the derivation of therapeutic procedures ( Rajewsky etਊl., 2020 ).

The First COVID-19 Vaccines: What’s mRNA Got To Do With It?

Most of us have an intuitive understanding of how a vaccine works: show the immune system a bit of a pathogen, or something mimicking it, and trick it into responding as if natural infection is happening. The COVID-19 pandemic ushered in a flood of vaccine options.

When I was writing &ldquoHow the various COVID vaccines work,&rdquo which ran here at DNA Science on September 10, I had to keep reviewing summary charts to remember who was doing what. Vaccine technology has gone beyond live, weakened, or killed virus, even past the once-groundbreaking subunit vaccines that present parts of a pathogen, like the hepatitis B surface antigen or pertussis toxin. Now we have DNA and RNA vaccines too, delivered in different ways.

The first two vaccines against COVID-19, Tozinameran (the Pfizer/BioNTech vaccine) and mRNA-1273, Moderna&rsquos still unchristened candidate on the brink of emergency use authorization, are mRNA. And that&rsquos confusing people, based, perhaps, on when they took high school biology (more on that coming). So here&rsquos a brief consideration of mRNA and how it can alert the immune system to fight SARS-CoV-2.

First, some things that the new vaccines are not and cannot do:
&bull They aren&rsquot viruses.
&bull They aren&rsquot &ldquonatural&rdquo &ndash they&rsquore synthesized.
&bull They can&rsquot enter a nucleus of a human cell and mutate our DNA. Even if they did, they&rsquod encode the viral spike protein &ndash as a vaccine does.
&bull They aren&rsquot derived from human embryos or fetuses.

Proteins lie behind traits, directly or indirectly. Clotting factors stop bleeding. Keratin forms hair and skin, collagen the body&rsquos glue, and hormones carry messages. Most of the enzyme types that propel metabolism fast enough for life are proteins (a few are RNA). Proteins pepper viral surfaces, like the trios of spikes from which the coronaviruses take their name and use to bind and invade our cells.

Genes consist of a sequence of DNA building blocks that form a code that tells cells how to make specific proteins, which are built of amino acids. Francis Crick described it in Nature in 1961. In translating the code from gene to protein, a cell transcribes the information into an intermediate form, to preserve the DNA database. That&rsquos messenger RNA, aka mRNA.

A cell makes mRNA for a simple reason: it can&rsquot use up its DNA and stay alive. Like a lone volume of a book in an old-fashioned library that someone takes out and never returns, DNA is precious. Copies ensure that the information persists, even if the original book or instructions vanish, and passes to the next cell generation during division.

Familiarity with mRNA Depends on Age

Some of the deer-in-the-headlights response to mRNA as a vaccine may come from members of the media who don&rsquot ordinarily report science, and oversimplify. CNN, for example, called RNA &ldquoa component of DNA.&rdquo No. Both are nucleic acids. (RNA differs from DNA in one of the four building blocks, RNA is single-stranded, and it&rsquos much shorter than DNA.)

When I began lecturing in an adult ed program, about 20 years ago, a gentleman took me aside during the break. &ldquoRicki, when these folks were in school, we didn&rsquot know about DNA.&rdquo That came in 1953 with Watson and Crick&rsquos famous paper.

Figuring out how genes encode proteins took longer, with experiments to crack the genetic code &ndash assigning RNA triplets to one of the 20 amino acids of proteins &ndash starting in 1959. &ldquoGenetic code&rdquo traditionally refers to that correspondence, and it is universal to all life and viruses. (See In Search of the Human Genetic Code).) More modern usage equates DNA, RNA, or genetic code with computer code RNA or DNA sequence is more accurate.

I learned about DNA, RNA, and protein &ndash the &ldquocentral dogma&rdquo of molecular biology &ndash in AP biology, then in college circa 1972. But the DNA-RNA-protein mantra has since migrated down to general high school biology, and I wrote about it in a middle school textbook. I imagine many people forgot the details as soon as the final exam was over, unless they took biology again in college. It&rsquos like my not knowing much about history.

But the central dogma is, well, central in high school biology. I worked on the New York State Science Learning Standards in 2016, which boil a very complex idea down: &ldquoConstruct an explanation based on evidence for how the structure of DNA determines the structure of proteins, which carry out the essential functions of life through systems of specialized cells.&rdquo

Modified mRNAs as Vaccines
An mRNA vaccine encodes a protein unique to a pathogen &ndash like a virus&rsquos spike. The two first COVID vaccines are gulped into cells and freed outside the nuclei, where the machinery that normally translates mRNAs into proteins goes to work. Soon, the cells release viral spike proteins &ndash but not viruses &ndash and cells of the immune system &ndash dendritic cells and macrophages &ndash sound the alarm. Within a few days, T cells activate B cells to crank out antibodies. Immunity has begun.

An mRNA vaccine can elicit a more powerful immune response than any other kind, but modifications improve on nature. A &ldquomodRNA&rdquo vaccine can evade the tiny bubbles of innate immune system proteins that can trigger potentially deadly overproduction of cytokines, and also avoid being chewed up by enzymes (ribonucleases).

The modified RNA &ndash something as simple as latching a methyl group (CH3) onto one of the four base types &ndash alters the encoded protein in subtle ways, modifying the twists and turns of the amino acid chain to fashion a topography that both shields the modRNA from the RNA-eaters, but allows production of spikes to proceed full speed ahead.

Additional modifications to parts of the mRNA that cap both ends further stabilize the molecules and boost spike protein output. And two amino acids &ndash prolines &ndash inserted at a key part in the sequence stabilize the spike protein in the three-dimensional shape that it naturally assumes just before it binds to the human ACE-2 receptors on cells.

The shots in arms happening all over the world right now represent decades of research.

A Brief History of mRNA Vaccines

Published reports of efforts to make mRNA vaccines go back to 1990 in mice and 1992 in rats. Much credit is now belatedly being given to Katalin Karikó, who sought grant funding to develop RNA-based vaccines starting in the mid-1980s. Her patent with co-worker Drew Weissman from the University of Pennsylvania, who is now with Pfizer partner BioNTech, was issued in 2006.

Tweaked, synthesized, modRNAs have been developed against Zika virus, influenza, cytomegalovirus, and two less familiar ones. So the stage was set when, on January 10, Chinese researchers published the first genome sequence of the new enemy, SARS-CoV-2.

The modRNA vaccines encode the 1273 amino acids that make up the viral spike protein &ndash hence Moderna&rsquos &ldquomRNA-1273.&rdquo (See COVID-19 Vaccine Will Close in on the Spikes,&rdquo posted here at DNA Science February 20, my third COVID article this one is #56).

I Failed at Finding the Recipes

I&rsquove read many articles and patents in search of the distinctions between the two mRNA vaccines. Here&rsquos Moderna&rsquos and here&rsquos Pfizer&rsquos.

Both vaccines encode spike protein with the added prolines. Both are delivered in lipid (fat) concoctions. Even those are similar. They consist of cholesterol, phosphocholine, polyethylene glycol, and the fourth is proprietary, a positively-charged special sauce. But the &ldquolipid nanoparticle&rdquo is just the carrier, melting into the cell membrane and shielding the RNA for a time inside the cell.

Both vaccines are engineered to be more visible to the immune system &ndash but is that due to the prolines, or an additional, proprietary tweak? Probably the latter, because something unique must distinguish the patents, and explain why the vaccines are not interchangeable. You can&rsquot start with Pfizer for shot one and switch to Moderna for shot two, or vice versa.

I read Moderna&rsquos 135-page protocol and patents, and entries for the COVID vaccines listed at, which currently number 328. The volume of information is stunningly overwhelming, as is the global effort to take down the minuscule monster that is SARS-CoV-2.

What I find most astonishing is that these viruses that slip inside our cells and do so much damage, even turning our immune systems against us in the molecular violence of a cytokine storm, can exert so much power because, ultimately, they came from our own genomes. They likely came from jumping genes, which are indeed a thing, discovered in the 1940s. How else could the viral spikes recognize and then swivel and bind to the molecules on our cells and invade?

What Causes Viral Infections?

Human cells are vulnerable to viruses, and when the body is exposed to viral particles, the immune system will try to destroy these particles and eliminate them from the system.

A lowered immune system allows the virus to more easily attach itself to available cells, often bringing about general symptoms such as fever, chills, and muscle aches. This also makes it easier for the virus to replicate, and thus advances symptoms until the immune system can fight the virus off.

Viral Infections in Children

Children often contract viral infections, as children spend time around other children who have colds, and this makes it more likely that the cold will be passed on to them. A child&rsquos immune system is not as strong as adults, and their body is still learning how to fight viruses for the first time. Some infections can become quite serious while some just bring about a feeling of unwell. Often children develop fevers, headaches, runny nose, cough, sore throat and fatigue. These symptoms are caused by the battle between the virus and the body's immune system.

  • It is important to allow babies and children to rest when they have a fever.
  • For younger children who cannot blow their nose, use a rubber suction bulb to suck drainage from both sides of the nose.
  • Loosen dried nasal drainage with warm water.
  • Children older than four years of age may be able to suck on throat lozenges for sore throats.
  • Children should drink extra water, fruit juices, or soups- avoid giving milk to infants for congestion reasons.
  • Use hot steam to loosen mucus in child&rsquos chest and nasal passages.

NOTE: See your doctor if high temperature persists for more than 5 days.

Viral infections and ME/CFS

A feeling of fatigue and exhaustion isn’t unusual in the aftermath of viral infections, but it usually passes. Accumulating evidence, however, suggests that in some patients viruses could be involved in triggering ME/CFS, a distinct clinical syndrome characterized by lasting fatigue that worsens after exercise or mental exertion—a hallmark physicians call post-exertional malaise. A light walk or completing a questionnaire can leave those with ME/CFS bedridden for days or even weeks.

See “Infographic: What is ME/CFS?”

“You don’t see that in any other condition,” says Alain Moreau of the University of Montreal who directs a research network for ME/CFS. “We have a large group of patients that are housebound. Even taking a shower could take hours, or they sometimes skip [it] because they cannot do it.” An inability to concentrate, or “brain fog,” is also common in the disease, adds Columbia University immunologist Mady Hornig.

The disease—formerly known simply as chronic fatigue syndrome, or CFS—has long been stigmatized to the point of being ignored by many physicians and researchers, in large part due to its mysterious etiology. Doctors would rule out a number of diagnoses, such as viral infections or neurological diseases, and conclude that there was nothing wrong with such patients, sometimes advising them to simply get more exercise, which would make their condition worse, notes Frances Williams, a genomic epidemiologist at King’s College London. An incident in which a high-profile study purported to identify definitive causes, which later turned out to be false, may also have discouraged scientists from studying ME/CFS, Nath adds. And while some drugs have been trialed in ME/CFS patients over the years, the results so far have been inconclusive, Moreau says, leaving few treatment options for the disease.

In part because of this long-term neglect of the disease, many patients prefer the term myalgic encephalomyelitis over chronic fatigue syndrome. ME implies a pathological process: an inflammation of the brain and spinal cord. However, Williams and Nath are quick to note that there is so far little evidence for encephalomyelitis in the condition, save for a small Japanese study that found elevated levels of inflammatory markers in the brains of ME/CFS patients and small changes in cytokines in their spinal fluid. Much of the research community, including the US Center for Disease Control and Prevention, have settled on calling the disease ME/CFS.

The question is not if [some] will develop ME/CFS—it’s how many.

Although it’s still a mystery what causes the disease, according to one survey, nearly 75 percent of ME/CFS patients have described viral infections prior to the onset of their symptoms. Other studies have linked particular pathogens, including West Nile, Ebola, and Epstein-Barr viruses, with the development of ME/CFS-like symptoms in substantial numbers of infected people. This association was also observed with SARS-CoV-2’s close relative, SARS-CoV, which caused the SARS epidemic of 2003. One study conducted a year after the SARS outbreak in Toronto found that fatigue was common among survivors, and 17 percent of them still hadn’t returned to work due to long-term health issues. Even three years after Toronto’s SARS outbreak, a study found widespread fatigue and achiness among those who had been infected.

Such findings leave Moreau with little doubt that SARS-CoV-2 could also leave some people with long-term disability, he says. “With this very severe COVID-19 disease, where we’re now dealing with millions of people suffering from it worldwide, the question is not if [some] will develop ME/CFS—it’s how many.”

Hornig notes that some of the long-hauler symptoms described in Davis’s report overlap with those common in ME/CFS, although only the passage of time will tell whether some long-haulers will meet the clinical definition of the disease. The rule of thumb is six months, Nath says, citing one of his own ME/CFS studies suggesting that patients rarely recover if their symptoms persist longer than half a year. He and others are now beginning to investigate not just whether, but how SARS-CoV-2 might lead to ME/CFS.


Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for a recent, unexpectedly severe epidemic in countries of the Indian Ocean region. Although many alphaviruses have been well studied, little was known about the biology and pathogenesis of CHIKV at the time of the 2005 outbreak. Over the past 5 years there has been a multidisciplinary effort aimed at deciphering the clinical, physiopathological, immunological and virological features of CHIKV infection. This Review highlights some of the most recent advances in our understanding of the biology of CHIKV and its interactions with the host.

Chikungunya fever, an arboviral disease that is caused by chikungunya virus (CHIKV) and is transmitted by mosquitoes, was first recognized in epidemic form in East Africa in 1952–1953 (Refs 1, 2). 'Chikungunya' is a Makonde word meaning 'that which bends up' and refers to the contorted posture of infected patients suffering from severe joint pain 3 . During the past 50 years, numerous CHIKV re-emergences have been documented in both Africa and Asia, with irregular intervals of 2–20 years between outbreaks 4 . The absence of serological surveillance means that precise numbers of individuals infected during these outbreaks can only be estimated. In 2004, CHIKV emerged in Kenya and spread to Comoros, where 5,000 cases were reported 5 . In 2005–2006, the outbreak spread to other islands in the Indian Ocean, including La Réunion this was the first time that CHIKV had infected an occidental country. La Réunion, which is part of France, is an island in the Indian Ocean with a population of ∼ 785,000 strikingly, an estimated 300,000 cases of CHIKV infections 5,6 and 237 resultant deaths 7 were reported. Viral genetic analysis supported the link between the infections in La Réunion and the outbreak in Kenya in 2004 (Refs 8, 9). The epidemic also spread to India, where it is estimated that more than 1.5 million people were infected, and it was subsequently identified in Europe and the United States, where it is thought to have been imported by infected travellers returning from areas with high incidence rates. Indeed, between July and September 2007 the virus caused the first autochthonous epidemic outbreak in the north-east of Italy, with more than 200 human infections all traced back to the same index case 10,11,12,13,14 . Currently, chikungunya fever has been identified in nearly 40 countries (Fig. 1), and in 2008 it was listed as a US National Institute of Allergy and Infectious Diseases (NIAID) category C priority pathogen 4,15 . Recent epidemic re-emergences were also documented in Kinshasa, the Congo (50,000 estimated cases in 1999–2000) 16 , Indonesia (2001–2003) 17 , the Indian Ocean islands of Mayotte, Seychelles, Mauritius and La Réunion 6 (300,000 cases in 2005–2006), India (1.4–6.5 million estimated cases in 2006–2007) 10,18,19 , and Malaysia and Thailand (3,000 and 42,000 estimated cases in 2009, respectively, according to the CDC), to name a few (Fig. 1).

Both blue and yellow indicate countries where cases of chikungunya fever have been documented, and blue indicates countries where chikungunya virus (CHIKV) has been endemic or epidemic. Figure is modified, with permission, from Ref. 4 © (2007) Society for General Microbiology.

CHIKV is a member of the family Togaviridae, genus Alphavirus 20 , which comprises enveloped, positive single-stranded-RNA viruses. In humans, CHIKV infection is of rapid onset and is typically cleared in 5–7 days. For reasons that are still being explored, the ongoing outbreak has been marked by severe symptoms 21 . The case fatality rate has been estimated to be 1 in 1,000, with most deaths occurring in neonates, adults with underlying conditions and the elderly 22,23,24,25,26,27 . Notably, these are the first documented deaths attributed to CHIKV infection.

In response to the public health need, and because La Réunion is a French territory, researchers in the Institut Pasteur, Paris, France, teamed up with clinicians in La Réunion and other countries to create a CHIKV task force. This multidisciplinary team has been working together for the past 5 years to analyse the epidemiology, physiopathology, virology, entomology and host response to infection. In this Review we highlight the recent major advances achieved not only by the CHIKV task force, but also by numerous other teams in the arbovirus community that have worked to address this important re-emergent infectious disease (see Box 1).

The genus Alphavirus contains approximately 30 members, which probably diverged a few thousand years ago 28,29 . Some alphaviruses are not pathogenic to humans, whereas others are highly infectious, with the associated clinical diseases ranging from mild to severe. Alphaviruses can be broadly divided into New World and Old World viruses 30,31 . These two groups have evolved distinct ways of interacting with their respective hosts and differ in their pathogenicity, tissue and cellular tropism, cytotoxicity and interference with virus-induced immune responses. It should be noted that most alphaviral infections in humans and domesticated animals are considered a 'dead end' — that is, the virus cannot be transmitted to a new host, so the evolutionary pressures driving viral diversification may be linked to their true host species. For CHIKV, a thorough exploration of other zoonotic viral reservoirs has not been carried out.

From a clinical perspective, the two groups of alphaviruses are subdivided into those associated with encephalitis (predominantly New World viruses) and those associated with polyarthritis and a rash (predominantly Old World viruses) 29,32,33 . Although CHIKV is a member of the arthritogenic alphaviruses, during the recent outbreak there were documented cases of meningoencephalitis (primarily in neonates) and haemorrhagic disease 22 , indicating that these signs are important sequellae of acute CHIKV infection 32,34,35 . Unlike typical encephalogenic alphaviruses, which infect neurons, CHIKV seems to infect the stromal cells of the central nervous system and, in particular, the lining of the choroid plexus (Fig. 2).

Transmission of chikungunya virus (CHIKV) occurs following a mosquito (Aedes aegypti or Aedes albopictus) bite. CHIKV then replicates in the skin, in fibroblasts, and disseminates to the liver, muscle, joints, lymphoid tissue (lymph nodes and spleen) and brain. The target cells are indicated for each tissue.

Transmission of CHIKV occurs through a bite by infected Aedes aegypti or Aedes albopictus , although in the recent epidemic some cases were the result of maternal–fetal transmission 22 . Following transmission, CHIKV replicates in the skin and then disseminates to the liver and joints, presumably through the blood 36,37,38 (Fig. 2). The incubation period is 2–4 days and is followed by a sudden onset of clinical disease with no prodromal phase (Fig. 3). Symptoms of CHIKV infection include high fever, rigors, headache, photophobia and a petechial rash or maculopapular rash. In addition, most infected individuals complain of severe joint pain that is often incapacitating 39,40,41 (see also the WHO guidelines on the clinical management of chikungunya). 'Silent' infections (infections without illness) do occur but are rare, being observed in around 15% of infected individuals 21 . Strikingly, during the acute phase, the viral load can reach 10 8 viral particles per ml of blood, and the plasma concentration of type I interferons (IFNs) is in the range of 0.5–2 ng per ml, accompanied by a robust induction of other pro-inflammatory cytokines and chemokines 42,43,44 (Fig. 3).

Following transmission by mosquito bite, infected individuals experience an acute onset of disease 2–4 days after infection. Symptoms include high fever, rigors, headache and a petechial or maculopapular rash. In addition, most infected individuals complain of severe joint pain that is often incapacitating. Disease onset coincides with rising viral titre, which triggers the activation of an innate immune response, the hallmark of which is the production of type I interferons (IFNs). Patients successfully clear the virus approximately 1 week after infection, and only at this time is there evidence of CHIKV-specific adaptive immunity (that is, T cell and antibody-mediated responses). Importantly, ∼ 30% of individuals experience long-term sequellae that include arthralgia and, in some cases, arthritis.

The acute phase of CHIKV infection typically lasts from a few days to a couple of weeks. In contrast to the acute phase, the chronic phase of disease has not been extensively investigated. Recurrent joint pain, which can last for years in some cases, is experienced by 30–40% of those infected, although this is not thought to be a result of chronic infection, as infectious virus cannot be isolated from these patients. Radiographic studies are typically normal or show mild swelling, which is consistent with joint pain. It has been suggested that this joint pain, similarly to the pain caused by the related alphavirus Ross River virus (RRV) 45 , is immune mediated. This has not been formally shown, although the presence of autoantibodies has been reported in one case of CHIKV infection with severe musculoskeletal complications 46 .

Cellular and tissue tropism

A large effort has been made recently to describe viral tropism and replication in cell culture systems and in animal models to better understand CHIKV pathogenesis (for details on the Alphavirus life cycle in mammalian cells, see Box 2). Studies in the 1960–1980s showed that CHIKV grows in a panel of non-human cell lines, including Vero cells, chick embryo cells, BHK21 and L929 fibroblast-like cells, and HEp-2 hepatic cells 47,48,49,50 . The cellular tropism of CHIKV in humans was characterized recently. In tissue culture experiments, the virus replicates in various human adherent cells, such as epithelial and endothelial primary cells and cell lines, fibroblasts and, to a lesser extent, monocyte-derived macrophages 51 . CHIKV also replicates in human muscle satellite cells, but not in differentiated myotubes 52 (Fig. 2). In contrast to adherent cells, B cells and T cells are not susceptible to CHIKV infection in vitro 51,53 . Like other alphaviruses, CHIKV is highly cytopathic in human cell cultures, and infected cells rapidly undergo apoptotic cell death 33,51 . This pattern of replication probably governs the pathological properties of the virus.

In a highly pathogenic mouse model in which animals lack the type I IFN receptor (Ifnar −/− mice) and are much more susceptible to severe disease, the CHIKV tissue tropism seems to match the tropism reported using in vitro systems. CHIKV was found to primarily target muscle, joint and skin fibroblasts, but it was also identified in the epithelial and endothelial layers of many organs, including the liver, spleen and brain 38 (Fig. 2). Notably, newborn and young mice are highly sensitive to CHIKV infection and represent a valuable model for studying CHIKV pathogenesis 38,54 .

Non-human primates have also been used as models for CHIKV-associated pathology and vaccine testing 55,56,57 . In two recent studies, intravenous or intradermal CHIKV inoculation of macaques resulted in high viraemia, peaking 24–48 hours after infection. Although infection was not lethal, it was associated with a transient acute lymphopenia and neutropenia (that is, loss of lymphocytes and neutrophils, respectively), an increase in monocytes and a pro-inflammatory response 56,57 . Infection recapitulated the viral, clinical and pathological features observed in humans 57 . CHIKV targeted lymphoid tissue, the liver, the central nervous system, joints and muscle during the acute phase 57 . Persistent infection (measured 44 days post-infection) occurred in splenic macrophages and in endothelial cells lining the liver sinusoids. Tissue derived from these animals carried low levels of replication-competent virus 57 . It will be important to establish whether this is reflective of the situation during human infection and what role viral persistence has in the chronic sequellae associated with chikungunya fever. One recent study has indicated that elderly patients are at high risk of chronic disease, but clearly more work is necessary 58 .

The human tissue culture systems and the simian and mouse models have provided clues about the tissue and cellular localization of CHIKV in infected humans. Samples from CHIKV-infected patients with myositic syndrome showed CHIKV antigen expression in skeletal muscle satellite cells but not in muscle fibres 52 . Infected fibroblasts have also been reported in biopsy material taken from acutely infected patients 38 . There is a debate about the sensitivity of primary blood monocytes to CHIKV infection 51,59 . Sourisseau et al. 51 reported that the high viral load in blood plasma (ranging from 10 5 to 10 8 RNA copies per ml) during acute infection does not correspond to detectable levels of viral RNA in blood cells. They also found that, in vitro, peripheral blood mononuclear cells (including B cells, T cells and monocytes) are not susceptible to CHIKV infection 51 . By contrast, Her et al. 59 observed that CHIKV antigens are detected in vitro in monocytes exposed to high viral inocula (multiplicity of infection = 10–50). CHIKV antigen-positive monocytes were also isolated from acutely infected patients 59 , but definitive evidence of productive infection was not established. As monocytes are phagocytic, and as viral titres are high in acutely infected patients, the presence of negative-strand viral RNA must be assessed to determine whether productive infection of monocytes does occur and whether monocytes are true targets of CHIKV. There are notable cell tropism variations among alphaviruses, which probably influences the pathogenesis of disease 30 . For example, human monocyte-derived dendritic cells (DCs) and plasmacytoid DCs (pDCs) are not sensitive to CHIKV 51,60 Venezuelan equine encephalitis virus (VEEV) can infect DCs and macrophages in lymphoid tissues and cultures, whereas this is not the case for Eastern equine encephalitis virus (EEEV) 61,62 . Interestingly, EEEV infection of myeloid-lineage cells is restricted after virus binding and entry, by inhibiting translation of incoming EEEV genomes 61 . Of note, RRV infects mouse macrophages 31,63,64,65 , which are implicated in the pathogenesis of disease. During RRV infection, infiltrates of inflammatory macrophages are observed in muscles and joints 45 , and treatment of mice with agents that are toxic to macrophages abrogated the symptoms of infection 66 .

The cellular tropism of alphaviruses is regulated by many parameters. For example, RRV envelope glycoproteins allow the infection of mouse DCs but not human DCs 67 , and the ability of Sindbis virus (SINV) 68 and VEEV 69 to infect DCs is determined by a single amino acid substitution in the E2 envelope protein. Further work should examine the sensitivity of Langerhans cells to CHIKV and other alphaviruses. The use of rhabdoviruses and lentiviruses pseudotyped with CHIKV envelope glycoproteins may facilitate the study of early entry or post-entry events 70 .

Type I IFNs (IFNα and IFNβ) are also major regulators of tissue tropism and virulence 71 . For example, they prevent the widespread dissemination of Semliki Forrest virus (SFV) in mouse extraneural tissues, and this is associated with reduced sensitivity to type I IFNs and enhanced virus pathogenicity 72 . More generally, type I IFN induction in vivo, as well sensitivity to type I IFN treatment in cell culture, differs markedly between different alphaviruses 73 . The interplay between CHIKV and the innate immune system is discussed below.

Jumping species — an atypical vector for CHIKV

CHIKV is endemic to Africa, India and Southeast Asia and is transmitted to humans by several species of mosquito, with geographical variations 33,74,75,76 . Although A. aegypti is the classical vector for CHIKV, the 2005 outbreak in La Réunion was associated with an atypical vector, A. albopictus 6,14,75,76,77,78 . Other Aedes species are sensitive to experimental CHIKV infection, but their role in field transmission has not been shown 79 .

Why did CHIKV adopt A. albopictus as its host? The transmission success of arboviral diseases depends on many factors, including the geographical and temporal distribution of the insect vectors, their growth rate and the viral incubation period inside them 80,81,82,83,84 . A. albopictus is a competent vector for dengue virus and numerous arboviruses, and its distribution has expanded recently, even replacing A. aegypti in some places 14,83,84,85 . It is native to Southeast Asia and has colonized both tropical and temperate regions. It was identified in Europe (first in Albania) and in North America in the early 1980s, probably having been introduced through shipments of used car tyres from Asia 86 . Currently, A. albopictus is present in at least 12 European countries and in around 25% of the United States.

There are several features of A. albopictus that make it a good viral vector: it survives in both rural and urban environments it was probably first zoophilic and then progressively became anthropophilic 87 it is long lived (4–8 weeks) it has a flight radius of 400–600 metres and it can successfully infect humans and animals because it is aggressive, quiet and diurnal. Furthermore, the mosquito's eggs are highly resistant and can remain viable throughout the dry season, giving rise to larvae and adults the following rainy season. All of these features of A. albopictus provided CHIKV with a great opportunity to infect humans once it had adopted this mosquito species as its host. In fact, the human–mosquito–human transmission cycle was so efficient that there was no identified animal reservoir during the epidemic in La Réunion 76 .

How was CHIKV able to efficiently adapt to A. albopictus? An extensive genomic analysis of recent clinical CHIKV isolates from the Indian Ocean outbreak identified unique molecular features when compared with the few previously available sequences from laboratory-adapted CHIKV 6 . In particular, changes were observed in E1 — a class II viral fusion protein that mediates viral entry at low pH 88,89,90 — potentially affecting viral fusion, assembly and/or cell tropism. Notably, a specific mutation in E1 (Ala226Val) was absent in the initial viral strains but was observed in >90% of the later strains 6 . Interestingly, in the related alphavirus SFV the amino acid residue at position 226 regulates cholesterol dependency during the virus–host cell fusion process 91 . The efficiency of alphaviral entry depends on host cell membrane composition (including the levels of cholesterols, which mosquitoes obtain through blood meals). A mutation that affects cholesterol dependency could improve the ability of CHIKV to infect insect cells by providing a better adaptation to the lipid composition of these cells. Indeed, experimental infection of A. albopictus showed that the early viral strains were not as successful at replicating in this mosquito as later, mutated viruses 75,76 . The E1 Ala226Val mutation is directly responsible for a substantial increase in CHIKV infectivity for A. albopictus and leads to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice 75 . Both early and late viruses invaded salivary glands in a similar pattern, but the crossing of the midgut epithelium, one of the primary sites of infection 75,76,92 , was a crucial step that made A. albopictus particularly susceptible to later CHIKV isolates 76 . Interestingly, this mutation has no effect on viral replication in A. aegypti 75 . Moreover, the E1 Ala226Val mutation facilitates viral replication in cholesterol-depleted C6/36 mosquito cells 75 . Other mutations that have been identified recently in E2 also regulate CHIKV adaptation to its mosquito hosts 93 . Whether the enhanced ability of later CHIKV isolates to invade A. albopictus relates to cholesterol dependency has not been proved yet, but these observations strongly suggest that the rapid evolution of CHIKV conferred a selective advantage on the virus to infect and replicate in A. albopictus. Of note, both early and late CHIKV isolates replicated similarly in various human cells 51 and in the non-human BHK21 cell line 75 .

In summary, the adaptive mutation of the virus to replicate in A. albopictus, which is more common than A. aegypti in some geographical regions and can act as an efficient vector for CHIKV, facilitated the spread of CHIKV. This, together with the fact that the human population had not previously encountered CHIKV and was therefore immunologically naive 84 , contributed to the magnitude of the La Réunion CHIKV epidemic.

Immune control of CHIKV

Epidemiological data from the CHIKV outbreak in La Réunion indicate that >85% of individuals harbouring antibodies for CHIKV reported symptoms of infection 21 . Although precise information regarding CHIKV transmission is difficult to obtain, the epidemiological data indicating that one-third of the island's inhabitants became infected suggest that CHIKV is highly successful. Humans, however, are not defenceless, and in fact CHIKV is efficiently cleared within 4–7 days of infection 94,95,96 (Fig. 3). As a typical adaptive immune response (for example, CHIKV-specific B cell and T cell activation) requires at least 1 week to develop, the innate immune system seems to be capable of controlling CHIKV. Below we discuss the innate and adaptive immune responses that are known to control CHIKV infection.

Innate immune control of CHIKV. From an immunological perspective, CHIKV and type I IFNs share a common history. Isaacs and Linemann 97 first described IFN as a substance with antiviral activity in 1957. CHIKV was discovered only 5 years earlier owing to a major chikungunya fever epidemic that lasted from the late 1950s to 1964 in Asia and South India 98 . It was at this time that the study of CHIKV intersected with the study of type I IFNs — in 1963, Gifford and Heller 99 reported in Nature that chick embryo fibroblasts infected with CHIKV produced detectable levels of type I IFNs 3 hours after infection. Despite a series of high-profile publications in 1963–1970 (including Refs 100, 101), the study of CHIKV was subsequently eclipsed by that of other model microorganisms.

Work over the past 50 years has defined type I IFNs as central to the control of viral infection. IFNα and IFNβ are mainly produced by leukocytes and fibroblasts, respectively. The production of type I IFNs is triggered by pattern recognition receptors (PRRs), which detect conserved molecular motifs — termed pathogen-associated molecular patterns (PAMPS) — including surface glycoproteins, single-stranded (ss) or double-stranded (ds) RNA and unmethylated CpG-containing DNA 102,103 . Two types of PRRs that recognize viral PAMPS have been identified: Toll-like receptors (TLRs which reside in the plasma membrane or the endosomal compartments) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs which reside in the cytoplasm) 104,105 . The TLRs comprise 11 transmembrane proteins, 6 of which (TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9) are known to be involved in antiviral immunity 106 . TLR2 and TLR4 can be activated by viral surface glycoproteins (for example, haemagglutinin of measles virus) 107,108,109,110 TLR7 and TLR8 are triggered by ssRNA (for example, that of influenza virus) 111 TLR3 is engaged by extracellular dsRNA 112 and TLR9 is activated by unmethylated CpG-containing DNA (for example, that of herpes simplex virus) 110 . RLRs include RNA helicases (such as MDA5 (melanoma differentiation-associated protein 5 also known as IFIH1), RIG-I and PKRs (dsRNA-dependent protein kinases) these detect viral RNA in the cytoplasm 113 . As CHIKV is a ssRNA virus that replicates with a dsRNA intermediate, potential sensors include TLR3, TLR7, TLR8 and the RLRs (Fig. 4).

Chikungunya virus (CHIKV) is a single-stranded RNA (ssRNA) virus and may generate double-stranded RNA intermediates during replication that have the potential to engage the pathogen recognition receptors Toll-like receptor 3 (TLR3), TLR7 and TLR8 and the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) melanoma differentiation-associated protein 5 (MDA5) and RIG-I. These receptors activate a signalling cascade that leads to the activation of type I interferons (IFNs) and the transcription of cytokines and chemokines. Recent evidence suggests that the production of type I IFNs by infected fibroblasts and other cell types is regulated by the adaptor protein CARDIF (CARD adaptor inducing IFNβ also known as MAVS), which acts downstream of MDA5 and RIG-I. The inflammasome may also induce IL-1β production by infected cells (not shown). In a mouse model, protection was also partly dependant on the TLR adaptor myeloid differentiation primary response protein 88 (MYD88). This may suggest a role for TLRs, possibly on haematopoietic cells. In addition, MYD88 also acts as an adaptor for interleukin-1β receptor (IL-1R), which could be activated by the secretion of IL-1β from infected cells, thereby inducing type I IFN in non-infected cells. IRF, IFN regulatory factor NF-κB, nuclear factor-κB TIR, Toll/IL-1 receptor domain TRAF, tumour necrosis factor receptor-associated factor TRIF, TIR domain-containing adaptor protein inducing IFNβ.

The mechanisms underlying type I IFN production following CHIKV infection were recently characterized. Previous data had shown that CHIKV does not directly infect primary leukocytes 51 , but it was expected that a ssRNA virus would be able to directly activate haematopoietic cells, especially pDCs. This assumption is based on the fact that pDCs express TLR7 and the observation that they can respond to viral PAMPs even in the absence of infection 114 . Remarkably, in vitro CHIKV infection of human peripheral blood mononuclear cells as well as of human and some mouse DC subsets indicate that this virus does not directly engage PRRs for the induction of type I IFNs 60 . Instead, using in vitro and in vivo studies, it was shown that type I IFNs are produced by infected fibroblasts 60 . The production of type I IFNs by infected fibroblasts is regulated by CARDIF (CARD adaptor inducing IFNβ also known as MAVS), which acts downstream of MDA5 and RIG-I, and may involve ssRNA detection by both RLRs (Fig. 4). On the basis of the CHIKV tissue tropism (Fig. 2), it has been argued that CARDIF is engaged in infected fibroblasts and stromal cells. However, adult Cardif −/− mice infected with CHIKV had only a subtle phenotype, suggesting that other sensors must also be involved in the host response to CHIKV. Indeed, in addition to induction by the RLR pathway, protection may also be mediated by myeloid differentiation primary response protein 88 (MYD88), which is an adaptor protein for several TLRs and for the interleukin-1β (IL-1β) receptor (Fig. 4). As Cardif −/− and Myd88 −/− mice were not as susceptible to CHIKV infection as Ifnar −/− mice, RLR and TLR recognition of CHIKV may cooperate for rapid clearance of the infection.

Two possible pathways may account for the role of MYD88 in the control of CHIKV infection. As stated above, haematopoietic cells are poorly stimulated by CHIKV, suggesting that the virus does not engage TLRs in a conventional manner 59,60 . There is, however, the possibility that endosomal TLRs are engaged as a result of haematopoietic cells phagocytosing infected cells, the latter being a source of viral PAMPs. For example, infection by SFV results in the generation of dsRNA that may engage TLR3 on CD8 + DCs following engulfment 115 . A second possible means of engaging MYD88 relates to its role as an adaptor for the IL-1β and IL-18 receptors 116 . There has been a surge of new information regarding the role of the inflammasome, which is well recognized as being crucial for IL-1β production following bacterial infection and also seems to participate in the control of viruses 117,118 . As such, IL-1β produced by CHIKV-infected cells following inflammasome activation may participate in viral control by stimulating non-infected cells in a MYD88-dependent manner 43,60 (Fig. 4).

The activation of PRRs triggers the production of type I IFNs, which are crucial for antiviral immunity. Indeed, mice lacking IFNAR are much more susceptible to severe chikungunya fever than wild-type mice 38 . Interestingly, using bone marrow chimaeras of wild-type and Ifnar −/− mice, it has been shown that type I IFNs mainly target non-haematopoietic cells, such as stromal cells, to achieve viral clearance 60 .

Type I IFNs, in turn, activate the transcription of interferon-stimulated genes (ISGs), as evidenced in infected humans, who have high levels of ISG products in the plasma 42 . ISGs contain promoter elements that are sensitive to interferon response factors (IRFs) 119 . There are >300 ISG proteins encoded in our genome and, although the function of most is unclear, those that are well characterized have been shown to have crucial roles in host defence 120 . The antiviral roles of ISG proteins have been defined for several related viruses. The most extensively studied is SINV, which can be controlled by RNase L 121 , ISG15 (Ref. 122), ISG49, ISG54, ISG56 (Ref. 123), ZAP (also known as ZC3HAV1) 124 and serpins 125 . For CHIKV, only one ISG involved in viral control has been defined so far: it has been reported that HeLa cells transfected with 2′, 5′-oligoadenylyl synthetase 3 (OAS3) are more resistant to CHIKV replication 126 . It remains unclear how OAS3 blocks CHIKV replication, but initial studies suggest that its function does not depend on its downstream effector, RNAse L 126 . Other ISG proteins are probably also involved in innate immune responses to CHIKV.

Similarly to other viruses, CHIKV is likely to have evolved mechanisms to modulate both the induction of type I IFNs and the effector molecules stimulated by type I IFN signalling pathways. On the basis of data from other Old World alphaviruses such as SINV and SFV, one candidate for this immune modulation is non-structural protein 2 (nsP2), which acts as an inhibitor of host protein synthesis 127,128 . Future studies will be required to decipher the crosstalk between the different ISGs involved in the innate immune response to CHIV and to determine the ISGs that are necessary for (as opposed to just capable of) inhibiting CHIKV replication.

Adaptive immune responses following CHIKV infection. Given the acute nature of CHIKV infection and disease pathogenesis, and the urgent need to tackle the spreading epidemic, there has so far been little effort afforded to understanding the sequellae of chronic infection with CHIKV and the role of the adaptive immune system in protection from subsequent re-infection. In fact, a deeper understanding of the humoral (that is, antibody-mediated) and cell-mediated immune response is important, as it is relevant for vaccine development and may impinge on our understanding of the chronic joint pain experienced by 30–40% of CHIKV-infected individuals.

One study showed that serum from donors in the convalescent phase contains neutralizing CHIKV-specific immunoglobulins 129 . Strikingly, it is possible to protect Ifnar −/− mice by administering these immunoglobulins, suggesting that sterilizing immunity is an achievable goal. Consistent with this interpretation, when CHIKV infection preceded the administration of CHIKV-specific immunoglobulins by 24 hours, the mice were no longer protected from lethal infection. Such passive immunity has been shown for other alphaviruses and may indeed be a viable medical intervention, especially in those individuals susceptible to severe CHIKV infection, such as neonates.

Even less is known about the role of lymphocytes during disease pathogenesis. One marked effect of CHIKV infection is acute lymphopenia. It has been reported that 80% of 157 individuals with acute CHIKV infection experienced a decrease in the frequency of circulating B cells and T cells. Nearly half of those individuals had lymphocyte levels that were one-quarter of the lower limit for healthy individuals 130 . This was probably not a direct effect of the virus on lymphocytes, as CHIKV does not infect B cells and T cells. Instead, it is possible that type I IFNs induce cell death in lymphocytes, as they do in other acute infections. In addition, upregulation of stromal IFN-stimulated chemokines (for example, CXC-chemokine ligand 10 and CC-chemokine ligand 5) can trigger the migration of lymphocytes from the blood to the tissues, leading to lymphopenia 131 . In most CHIKV-infected individuals, repopulation of the circulating pool of lymphocytes occurs soon after resolution of infection. Interestingly, RAG-deficient mice (which lack lymphocytes) can clear CHIKV infection (C. Schilte & M.A., unpublished observations), suggesting that lymphocytes are not crucial for immunity during acute infection. However, this observation must be interpreted with caution, as mice are not the natural hosts of CHIKV. Nonetheless, the kinetics of viral clearance and the absence of data regarding exacerbated disease in humans with weakened adaptive immunity (for example, individuals infected with HIV) suggest that the innate arm of the immune response is sufficient for clearance of the infection in humans as well.

The role of cytotoxic T lymphocytes (CTLs) in particular during alphavirus infection has barely been studied so far. A dominant mouse CTL epitope present in a conserved region of the capsid of Old World alphaviruses has been described 64 , strongly suggesting that CTLs can be induced by CHIKV. Whether CTLs participate in the elimination of CHIKV-infected cells in humans remains to be addressed.

One side effect of adaptive immune responses is the possible induction of autoimmunity, caused by cross-reactivity between viral and host antigens. Again, there is little information on this subject, but there is certainly a possibility that B cell and T cell responses to CHIKV are implicated in the long-term joint disease experienced by many convalescent patients 46 . More information and careful epitope mapping are needed to determine whether some of the clinical findings of CHIKV infection are caused by autoimmune reactivity.

Vaccine development. The initiative to stimulate protective immunity as a strategy for preventing CHIKV infection in humans began in the early 1970s. Two formulations showed early promise: formalin fixation and ether extraction were both successful means of inactivating CHIKV while maintaining its ability to stimulate the production of haemagglutination-inhibiting, complement-fixing and neutralizing antibodies 44,132 . These initial studies included human trials, with 16 army recruits receiving formalin-fixed CHIKV vaccine prepared in bank-frozen green monkey kidney tissue culture 132 . Work progressed slowly, but the US Army remained committed to this effort and in 2000 carried out a Phase II clinical trial examining the safety and immunogenicity of the use of live attenuated CHIKV vaccine 55,133,134 . A 1962 strain of CHIKV from an outbreak in Thailand was used in this case, and the vaccine was formulated as a lyophilized supernatant from human MRC-5 cells. Of the 58 study subjects that received the vaccine, all developed neutralizing antibodies, and 5 subjects experienced mild to moderate joint pain 134 .

One important issue that arose during these early studies is the potential interference arising from sequential administration of vaccines specific for heterologous alphaviruses. Specifically, individuals vaccinated against VEEV showed poor neutralizing-antibody responses to the CHIKV vaccine 133 . Similarly, vaccination with CHIKV followed by VEEV resulted in reduced VEEV-specific responses 133 . This is a concern, as the populations at risk for these agents live in overlapping geographical regions.

Following the recent epidemic, there has been a renewed effort for vaccine development. A new formulation using virus-like particles has been shown to induce neutralizing antibodies in macaques 56 . These antibodies offered protection following challenge with different strains of CHIKV, and transfer of the macaque antisera into highly susceptible Ifnar −/− mice protected the mice from infection 56 . This approach may prove useful, not only for vaccination against CHIKV, but also for vaccination against other pathogenic alphaviruses.

An unprecedented effort teaming up clinicians, virologists, immunologists, molecular biologists and entomologists throughout the world has considerably furthered out understanding of CHIKV biology. Viral replication has been extensively studied in mammalian and insect cell culture systems. Biological samples from acutely and chronically infected humans have been analysed and, together with the development of animal models, have provided invaluable tools for studying the physiopathology of infection. CHIKV shares many characteristics with other Old World alphaviruses but also displays unique and previously unexpected properties.

Important questions remain to be addressed. The relative roles of the virus and the immune system in acute and chronic pathologies associated with CHIKV infection have yet to be deciphered. Analysing the impact of the adaptive immune response on controlling infection will have implications for the development of vaccine strategies. At the cellular and molecular levels, identifying additional members of the array of sensors involved in viral detection will bring new insight into the interaction of the virus with the innate immune system. From a virological standpoint, the role of non-structural viral proteins as well as the identity of cellular receptors allowing viral entry are partly unknown.

Perhaps one sad reality that we must reflect on is that CHIKV research received so much support as a direct result of the epidemic having emerged in an occidental country — an island that is part of France. Weekly articles in the lay press documented the escalation of cases during 2005 and 2006, as well as the deaths in infected neonates. Our awareness of the disease (and the real possibility of there being a worldwide problem) was increased by the reports of primary infections in Italy during the summer of 2007. Nonetheless, more needs to be done to educate the public about the risks associated with re-emergent viruses such as CHIKV. Clearly, a virus capable of infecting an estimated 7.5 million people over a 5-year period, resulting in chronic arthralgia in ∼ 30% of these individuals, deserves more attention. Private and public funding organizations have helped to raise awareness for global health issues such as HIV infection, malaria and tuberculosis, but this unfortunately represents only a proverbial 'small bite' out of a major problem.

Box 1 | A multidisciplinary approach to addressing public health problems

The chikungunya virus (CHIKV) outbreak in La Réunion highlighted the importance of using a multidisciplinary approach to address medical and public health issues. Numerous teams in the arbovirus community have rapidly focussed their studies on CHIKV. One noticeable initiative was the creation of a CHIKV task force comprising virologists, epidemiologists, entomologists, pathologists, immunologists and clinicians working in La Réunion. Epidemiologists and virologists defined new mutations that emerged during the 2005–2006 outbreak. Clinicians from several medical specialties studied the clinical features of infection in neonates. Entomologists and virologists characterized the vector change from Aedes egyptii to Aedes albopictus. All of the groups worked together to define viral tropism in humans and animal models. This progress, together with a collaboration with key industrial partners, resulted in the development of new tools for the diagnosis of CHIKV infection and the availability of new information regarding the treatment and management of individuals susceptible to severe disease.

Such multidisciplinary efforts are uncommon in life sciences. A major barrier to the multidisciplinary approach is the specialization required to train highly focused professionals, who in turn often construct a 'boundary' around their area of expertise. This has resulted, in many cases, in the development of jargon that cannot be understood by other scientists, even those in closely related fields. Although this 'specialist' approach has led many technological and conceptual advances over the past 50 years, it could be argued that the complex nature of disease pathogenesis requires a team-based approach to problem solving. In addition, the advent of '-omics' research has resulted in the generation of a plethora of data that cannot be integrated and applied by an individual research unit. Physicists, chemists and even clinicians have all understood the need for collaborative, multidisciplinary approaches. The rapid response to the CHIKV outbreak on the part of the scientific community shows that the power of collaborative science can extend to public health and life sciences. This was only a first step, as additional work will be required to identify new treatments and prophylactic strategies against this pathogen.

Box 2 | The alphavirus life cycle

The Alphavirus life cycle is depicted in the figure. Alphaviruses enter target cells by endocytosis 33 . A few receptors (for example, dendritic cell-specific ICAM3-grabbing non-integrin 1 (DC-SIGN also known as CD209), liver and lymph node-SIGN (L-SIGN also known as CLEC4M), heparan sulphate, laminin and integrins) have been implicated in this process, but their precise roles have not been firmly established 33 . Following endocytosis, the acidic environment of the endosome triggers conformational changes in the viral envelope that expose the E1 peptide 90,135 , which mediates virus–host cell membrane fusion. This allows cytoplasmic delivery of the core and release of the viral genome 6,29,136 . Two precursors of non-structural proteins (nsPs) are translated from the viral mRNA, and cleavage of these precursors generates nsP1–nsP4. nsP1 is involved in the synthesis of the negative strand of viral RNA and has RNA capping properties 33,137 , nsP2 displays RNA helicase, RNA triphosphatase and proteinase activities and is involved in the shut-off of host cell transcription 138 , nsP3 is part of the replicase unit and nsP4 is the viral RNA polymerase 33 . These proteins assemble to form the viral replication complex, which synthesizes a full-length negative-strand RNA intermediate. This serves as the template for the synthesis of both subgenomic (26S) and genomic (49S) RNAs. The subgenomic RNA drives the expression of the C–pE2–6K–E1 polyprotein precursor, which is processed by an autoproteolytic serine protease. The capsid (C) is released, and the pE2 and E1 glycoproteins are generated by further processing. pE2 and E1 associate in the Golgi and are exported to the plasma membrane, where pE2 is cleaved into E2 (which is involved in receptor binding) and E3 (which mediates proper folding of pE2 and its subsequent association with E1). Viral assembly is promoted by binding of the viral nucleocapsid to the viral RNA and the recruitment of the membrane-associated envelope glycoproteins. The assembled alphavirus particle, with an icosahedral core, buds at the cell membrane.

What’s Wrong with Human/Nonhuman Chimera Research?

The National Institutes of Health (NIH) is poised to lift its funding moratorium on research involving chimeric human/nonhuman embryos, pending further consideration by an NIH steering committee. The kinds of ethical concerns that seem to underlie this research and chimera research more generally can be adequately addressed.

Citation: Hyun I (2016) What’s Wrong with Human/Nonhuman Chimera Research? PLoS Biol 14(8): e1002535.

Published: August 30, 2016

Copyright: © 2016 Insoo Hyun. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The author received no specific funding for this work.

Competing interests: The author has declared that no competing interests exist.

Abbreviations: ISSCR, International Society for Stem Cell Research NIH, National Institutes of Health

Stem-cell–based human/nonhuman chimera research involves the transfer of human stem cells into animal hosts at various stages of development. The purpose of this research is to introduce localized human cellular and biological characteristics into laboratory animals to advance stem cell science, developmental biology, and many areas of biomedicine. Human/nonhuman chimera research has existed without much controversy for decades outside of stem cell research, resulting in, for example, mouse models of human cancer and the human immune system [1]. However, the possibility of acute levels of human/nonhuman mixing in stem-cell–based chimeras seems to be of special concern to many, as I discuss in this Perspective piece.

The debate over stem-cell–based chimeras is no mere philosophical quibble: it has real, practical import for research funding. Chimera research was thrust into the spotlight in September 2015 when the National Institutes of Health (NIH) announced a moratorium on the funding of research in which human pluripotent stem cells are transferred into nonhuman vertebrate pre-gastrulation stage embryos ( Now the NIH has announced it might allow public funds for this type of research under a proposed policy that would utilize input from a steering committee established to assess and oversee human/nonhuman chimera research ( Until this new approach is finalized, however, the current moratorium will remain in place.

Interestingly, the NIH moratorium does not include other forms of human/nonhuman mixing that would seem to be in the same ethical ballpark as human stem cell transfers into animal embryos, such as genetic humanizations of mice [2] and human glial progenitor cell transplantations into neonatal mouse brains [3]. One may wonder why this is the case.

The NIH’s funding moratorium appears to have been triggered by a branch of stem cell research that aims to grow transplantable human organs in genetically modified large animals, such as pigs and sheep. Researchers have shown that it is possible to grow a rat pancreas in a mouse and vice versa by “complementing” the preimplantation embryo (blastocyst) of one species with pluripotent stem cells of the other once the embryo has been genetically modified in vitro to lack its own pancreas [4]. Building on the success of these techniques, researchers are now interested in complementing pancreatogenesis-disabled pig embryos with primitive, so-called “naïve” human pluripotent stem cells for a similar purpose: to grow a human pancreas in a pig [5]. This line of research, which is currently being supported with nonfederal funds, could open the door to growing various types of human organs such as hearts and kidneys in livestock animals for transplantation in the future. If the pluripotent stem cells for transfer are created using a patient’s own tissue sample, then, in theory, the resulting organ would be immune-compatible with the patient. The humanitarian importance of this research is both apparent and urgent. There is currently a dire shortage of organs for transplantation in the United States, leading to approximately 22 deaths per day among patients waiting for organs.

Given the noble aims of this research, it is puzzling to some why the NIH is so nervous about providing federal funds to researchers with a track record of success in this area. The NIH has for years supported research in which human cells are transplanted into animal models, and it continues to fund human/nonhuman chimera research that lies outside the scope of research singled out in its notice of moratorium. How might this current policy difference be explained?

One possible answer is that there may be unique ethical risks involved in growing human organs in animals. In particular, some might worry that embryo complementation could inadvertently affect the developing animal in ways that go well beyond the localized formation of human organs, resulting in an acutely chimeric animal with an ambiguous moral status. Apparently, the threat of such an outcome simply may not be held to be as present in non-stem-cell–based chimera research. The transfer of cancerous human cells into postnatal immune-deficient rodents or the swapping of a mouse’s immune system with a human one arouses little ethical concern because these forms of chimerism are limited to more mature cell types and anatomical sites that have no obvious bearing on an animal’s moral status.

Unlike these other examples of human/nonhuman chimerism, stem-cell–based chimerism has the potential to radically humanize the biology of laboratory animals, depending on the type and number of human stem cells transplanted, the species and developmental stage of the host animal, and the anatomical location of the animal host where the human stem cells are transferred [6]. When human stem cells are transplanted into a postnatal animal, it is unlikely these cells will integrate significantly into the animal’s existing biological structures. But if human stem cells are introduced into an embryonic or fetal animal host that is then gestated, then the fractional percentage of differentiating human cells and the degree of human physiological integration in the developing chimeric animal may turn out to be high, especially if there is little evolutionary distance between the animal species used and humans. The worry, therefore, is that in the process of biologically humanizing a research animal, scientists may end up also morally humanizing the resulting chimera, especially if there is acute human/nonhuman chimerism of the central nervous system.

It is easy, however, to overstate the concern about the moral humanization of acute human/nonhuman chimeras, and several considerations should serve to turn down the heat around this speculative concern. First, the interspecies boundary that exists between humans and livestock is sufficiently high that it is quite unlikely that acute chimerization of all aspects of the resulting animal will occur. As a case in point, the mouse/rat chimeric experiments produced about 20% donor chimerism on average outside the relevant organ niche. The interspecies barrier is far lower between these rodents than it is between humans and livestock, thus making the degree of possible “off-target” human chimerism much less worrisome in the host animal. (Indeed, the evolutionary distance between pig and human is actually greater than the distance between human and mouse.) Second, in order to ensure even further that livestock blastocyst complementation using human stem cells avoids unwanted chimerism of the animal, especially within the central nervous system, researchers are now developing what they call “targeted organ generation” [7]. Here, the transferred human stem cells would be genetically modified to differentiate down only the endodermal lineage that produces the organs of interest, thus preventing the possibility that they would develop into human neural cells, which are derived from the ectodermal lineage. And third, researchers can take care to proceed in a stepwise fashion through a series of pilot studies, stopping their chimera experiments each time well before the full gestation cycle to examine fetal tissues for any unwanted migration and development of human cells outside the organ niche environment.

The precautionary steps outlined above are consistent with the ethical standards for chimera research set forth by the Ethics and Public Policy Committee of the International Society for Stem Cell Research (ISSCR), which was offered as a resource for the global stem cell community in 2007 [8]. This advisory report builds its recommendations on top of animal welfare principles that are already in play for the use of genetically modified animals, which include: (1) the establishment of baseline animal data (2) ongoing data collection during research concerning any deviation from the norms of species-typical animals (3) the use of small pilot studies to ascertain any welfare changes in modified animals and (4) ongoing monitoring and reporting to oversight committees. In addition to these four conditions, the ISSCR Ethics Committee recommends that ethical assessments of chimera research should be based on rational, practical, fact-based assessments of the developmental trajectories that are likely to be affected, taking into account the biological context in which the human cells are going to be deployed. More attention needs to be placed on animal welfare standards, especially as they pertain to livestock animals for research, and less focus should be placed on speculative concerns about the “moral humanization” of chimeric animals. I conclude with a few words on this last point.

If by “moral humanization” one means the appearance of uniquely human psychological characteristics in a chimeric animal, then two considerations expose the unrealistic assumptions undergirding this concern. The first consideration is that it is entirely unclear what types of new psychological characteristics could count to elevate the moral status of a research animal above where it currently is, such that its scientific use would no longer be morally acceptable. In my view, the only characteristic that might qualify doing this heavy moral lifting is the appearance of human-like self-consciousness, defined as an existential awareness and concern for oneself as a temporally extended agent with higher-order beliefs about one’s own mental experiences. But this unique psychological characteristic is not likely to emerge in a chimeric animal’s brain, as it takes several years to develop in infant brains that are 100% human and only under the right social and nurturing conditions of child-rearing [9]. The second consideration is that people tend to assume the presence of human cells in an animal’s brain might enhance it above its typical species functioning. Yet this seems to be an extreme form of anthropocentric arrogance—an unstated moral imperialism connected to human stem cells—to assume that the presence of human neural matter in an otherwise nonhuman brain will end up improving the animal’s moral and cognitive status. The much more likely outcome of neurological chimerism is not moral humanization of the animal in this sense but rather an increased chance of animal suffering and acute biological dysfunction and disequilibrium, if our experience with transgenic animals can be a guide. This is why the ethics and regulation of chimera research should prioritize animal welfare principles while at the same time enabling scientific progress in areas of humanitarian importance, albeit in a manner consistent with these principles.

In this Perspective piece, I have attempted to lay bare the sort of concerns that appear to underlie the NIH’s moratorium on chimera research funding. I suggest that most, if not all, of these concerns can be reasonably addressed. Although stem-cell–based chimera research encompasses a wide array of research activities, the issues surrounding the NIH moratorium nicely encapsulate the ethical concerns that are common across many forms of chimera research. Thus, my analysis in this Perspective might provide an efficient way to target the ethics of chimera research more generally.

Why Revive a Deadly Flu Virus?

One morning last August, Terrence Tumpey, a research scientist at the Centers for Disease Control and Prevention in Atlanta, walked into a room across a corridor from his office and took off all his clothes. He pulled on cotton scrubs and a disposable gown, two pairs of latex gloves and headgear with a clear plastic shield enclosing his face and a tube running out the back to a set of filters strapped to his waist. He walked through another door and down a hallway to a large upright freezer. Mounted beside the freezer was a retinal scanner. Tumpey, who is 6 feet tall, bent down a little to position his eyes in line with the lens. In a digital voice, the scanner asked him to step forward. Tumpey complied. "Identification confirmed," the scanner said, and a lock on the freezer clicked open.

Inside the freezer were trays and boxes containing "select agents" -- highly pathogenic microbes that under the Patriot Act cannot be handled without special clearance from the Department of Justice. Tumpey wiped the frost off a box. He was the only person in the C.D.C., or anywhere else, authorized to handle this particular agent: a synthesized version of an influenza virus that, nearly a century before, killed between 20 million and 50 million people. He placed the box in a secure container, and after showering and dressing, carried the container through secure corridors to another building at the C.D.C., where he entered another suite of rooms, dressing once again according to Biosafety Level 3+ protocols, the second most stringent level of biosecurity. For the next couple of hours, he squirted the virus into the nostrils of laboratory mice. He was fairly certain they would all soon die.

Getting the flu can be a real drag. Your head pounds, your muscles ache, you lie in a bed of misery, surrounded by clammy tufts of used Kleenex you're too tired to pick up. Every year, 5 to 20 percent of the American population catches a flu virus. The elderly, very young children and people with certain health conditions are at risk for more serious complications, and annually some 36,000 of them die. Every few decades, a particularly virulent strain appears and causes a global pandemic. In the 20th century, flu pandemics occurred in 1918, 1957 and 1968. The last two killed two million and 700,000 people respectively -- again, claiming most of their victims among the young, the old and the weak.

The 1918 flu virus is remarkable for two reasons. First, it caused perhaps the most lethal plague in the history of humankind. In the fall of that year it spread across the planet, perversely striking down healthy young adults. Once ensconced in their lungs, the virus triggered a havoc of inflammation, hemorrhage and cell death. Trying to draw air into such lungs was like breathing through meat. Many of the afflicted died within hours after they first began to feel a little feverish. Others succumbed more slowly to secondary bacterial infections. By the spring of the following year, the virus had disappeared as mysteriously as it had come.

The second, and in some ways even more remarkable, thing about the 1918 flu virus is that it has literally been brought back to life. In October, a team of scientists, Tumpey among them, announced that they had recreated the extinct organism from its genetic code -- essentially the scenario played out in the movie "Jurassic Park," albeit on a microbial scale. In the movie, the scientists' self-serving revivification of dinosaurs leads to mayhem and death. Tumpey and his colleagues say they hope that their resurrected microbe will help prevent a calamity, not cause one. They want to know what made the 1918 flu, which began as a virus native to wild birds, mutate into a form that could pass easily from one human to another. That question has been weighing on the minds of flu experts since 1997 -- since the first fatal case in Hong Kong of the avian flu that has since killed more than 70 people in Asia. So far, all of its victims probably caught the disease from handling infected poultry and not from other people. How close is it to crossing the same lethal line that the 1918 virus did? What can be learned from the virus that caused the great pandemic that might help us avert another one?

The risks involved in trying to answer such questions are hard to calculate, because the experiment has no precedent. In essence, Tumpey and his colleagues have brought one serial killer back from the grave so that it can testify against another. How dangerous is the 1918 virus to today's population? Its genetic code is now in public databases, where other researchers can download it to conduct experiments. Scientists from the University of Wisconsin and the National Microbiology Laboratory in Canada have already collaborated to reconstruct the virus from the publicly available sequence. How easy would it be for a bioterrorist to exploit the same information for malevolent ends?

"Give me $100,000 and two months, and I can recreate it right here in my lab," says Earl Brown, a flu researcher who specializes in the evolution of virulence at the University of Ottawa. "You wouldn't be able to tell it from the real thing that was around a hundred years ago. Would it kill at the same rate as in 1918? Probably. But you really don't want to have to find that out. You don't want to give this thing a second time around."

Terrence Tumpey is not moved by such talk. Even if the virus was to get out into the population, he says he believes it would cause far less sickness than it did in 1918. And he is sure that it is not getting out, ever, at least from his lab at the C.D.C. But whatever the danger posed by the virus in his freezer, it is literal living proof that science has crossed into an uncertain new world, where the drive to know life on its most fundamental level has given birth to the means to create it.

The resurrection of the 1918 influenza virus was a team effort engaging the resources of the C.D.C. in Atlanta, an obscure military pathology lab outside Washington, D.C., an esteemed group of influenza experts at Mount Sinai School of Medicine in New York and one elderly Swede. Though the story has been told before, it is impossible not to begin with the Swede. In 1950, Johan Hultin, then a 25-year-old graduate student at the University of Iowa, was searching for a Ph.D. topic when he heard a visiting virologist say that the only way to solve the mystery of the 1918 pandemic would be to recover the virus from a victim who had been buried in permafrost. Hultin suddenly had a topic.

After some planning, he found what seemed like an ideal site in the remote settlement of Brevig Mission on Seward Peninsula in Alaska. In a mere five days in November 1918, 72 of the 80 residents of Brevig died and were later buried in a mass grave. Hultin arrived there alone, obtained permission to dig up the grave and after two days of hacking through frozen ground came across the preserved body of a little girl in a blue dress, red ribbons in her hair. He and some colleagues eventually found four more bodies and cut out samples of their pocked and peppered lungs, keeping them frozen with dry ice exuded from fire extinguishers.

Back in Iowa, Hultin injected a solution of the lung tissue into fertilized chicken eggs -- a standard method for growing flu virus -- and inoculated mice, rats and finally ferrets, which have a peculiar susceptibility to human flus. Nothing worked. If the virus was there at all, it was dead. So was Hultin's Ph.D. thesis. He gave up, went to medical school and enjoyed a successful career as a pathologist in San Francisco. In his spare time he traveled all over the world, invented auto-safety equipment, restored archaeological sites, built a replica of a 14th-century Norwegian cabin in the Sierras (it took him 36 years) and did research on Mount Everest. But he never forgot about the one time in his life that he failed.

Jeffery Taubenberger, the man most responsible for resurrecting the 1918 flu virus, was looking a little sick. His face was pale and his eyes red-rimmed, and he had barely touched the pasta he ordered for lunch. He pulled out a handkerchief and sneezed hard.

"There's not a respiratory virus on earth that I don't seem to want to amplify," he told me. "If I were alive in 1918, Iɽ be dead."

Taubenberger is the chairman of the department of molecular pathology of the Armed Forces Institute of Pathology in Rockville, Md. His department was, in the early 90's, in the process of developing an expertise in retrieving tiny whispers of genetic code from putrefied flesh. As Gina Kolata described in her book "Flu," Taubenberger decided in 1995 to look for the 1918 virus in samples of preserved lung in the A.F.I.P.'s tissue repository, which contains about three million pathological samples dating back to the Civil War. His techniques were far more advanced than anything Hultin had at his disposal, and his goal was more modest. Taubenberger knew that flu particles are too unstable to remain intact in a frozen corpse, and he wanted only to find a remnant of the virus's genetic code, perhaps enough to reveal what made it so virulent. But for a year and a half, he, too, failed. Finally, when Taubenberger was on the verge of giving up, he recovered from a soldier's lung a tiny fragment of the killer flu's identity, like the upturned edge of a sneering mouth.

"From that moment on, I became the steward of this virus," Taubenberger said. "Whether I liked it or not, I was obligated to get the whole thing."

Taubenberger is a compact, attractive man in his mid-40's, with big, dark eyes and a quiet, precise manner of speech. He looks a bit like Frodo the hobbit in the movie version of "The Lord of the Rings," if you can imagine a middle-aged Frodo wearing a paisley tie and an oxford shirt, a cellphone strapped to his belt. Like Tolkein's hero, Taubenberger seems both obsessed with his quest and a little tired of shouldering its weight. The trace of the virus in the soldier's lung was unimaginably faint. But by using what is called the polymerase chain reaction (P.C.R.), a common method of amplifying a signal of DNA in a sample, he and his colleague Ann Reid were able to fish out a strand large enough to sequence then they used that sequence as a hook to fish out another strand, then another, gradually overlapping pieces that matched on their ends to build increasingly longer and more coherent pieces.

"We had to tweak the P.C.R. method to its ultimate level of detection," Taubenberger said. "It wasn't simple. It was painful. Everything we did here was painful."

Almost immediately he and Reid ran into another problem: they were running out of raw material. Then, out of the blue one day in 1997, he got a letter. It was from Johan Hultin, then 72, who had read about Taubenberger's initial success in Science magazine. He told Taubenberger about his expedition to the mass grave in Brevig in 1951 and said he would be willing to go back and try to find the virus again. Hultin said he would pay for the expedition himself. If he failed, no one else need know that it had ever happened.

And that is how Johan Hultin returned to Brevig -- a tall, gray-bearded figure arriving unannounced, carrying his wife's pruning shears to help him cut through bone. After again obtaining permission, he reopened the grave, and on the fourth day of digging found the body of an obese woman whose lungs were well preserved, insulated from the occasional ground thaw by her fat. He returned home with samples of her lungs and other organs and sent them to Taubenberger. The entire expedition took five days.

"Ten days later, he called me," Hultin said of the conversation with Taubenberger. "I was in my Norwegian cabin in the mountains. 'We have the virus,' he said. Iɽ been waiting 50 years to hear that."

A flu particle is a sphere about a millionth of an inch in diameter, containing just eight disconnected gene segments. Its surface is covered with a thicket of spikes, like a burr. The spikes are made of a protein called hemagglutinin, which sticks to receptors on the surface of cells in your respiratory tract, much as the hooked spines on a burr catch fast on fibers in your trouser leg when you're walking through high brush. In among the spikes are some other, mushroomlike protrusions of another protein, neuraminidase. These two surface proteins define the virus's identity -- the face that your immune system sees and attacks. Sixteen "flavors" of hemagglutinin are known, and nine of neuraminidase. The different major families of flu are combinations of the two, hence the designation "H5N1" for the current threat. The 1918 virus was H1N1, the mother of all flus.

Flu viruses mutate very rapidly, and each season's version is a little different. But your immune system preserves a memory of its previous encounters with a flu, which are dragged up, like old photographs from the back of a closet, every time your system responds to a new flu invasion. Very rarely, a virus comes along bearing a surface protein that your immune system has never seen. Often this occurs when a single host -- it could be a pig, but might also be a person -- becomes infected with two strains of flu simultaneously, one from a mammalian lineage, the other from an avian one. Inside the host, the eight gene segments of the two strains are shuffled randomly into new configurations, like the symbols in the window of a slot machine. If one of these configurations happens to be both pathogenic and transmissible from human to human, jackpot: a pandemic ensues. The 1957 and 1968 pandemics both probably occurred through this kind of "reassortment." For a long time, most scientists believed the same kind of gene-shuffling triggered the far more calamitous 1918 pandemic as well.

In his hunt for the cause of the 1918 flu's virulence, Taubenberger focused first on the hemagglutinin gene. Seasonal flus are normally confined to the respiratory tract because before it can infect a cell, the hemagglutinin protein needs to be split down the middle by an enzyme found there. But some forms of avian flu -- including H5N1, the one now threatening us -- bear a specific mutation in their hemagglutinin gene that allows other, more ubiquitous enzymes to cleave apart the protein, freeing the virus to invade cells deeper in the lungs or even in other organs. Taubenberger looked for the same killer mutation in the 1918 virus's hemagglutinin gene, but it wasn't there. After months of more work, he and Reid decoded the gene for neuraminidase. It, too, gave no hint why this particular virus was so deadly.

Same for the next gene, and the one after that. A year went by, then another. Instead of revealing some peculiar feature that might tip off the secret of its virulence, the genetic sequence of the virus slowly emerging seemed chillingly ordinary. Among the chain of some 4,000 amino acids that made up its proteins, only 25 or 30 distinguished it from a common, nonvirulent avian flu. Rather than originating from a reassortment of genes from both an avian and mammalian source, like the viruses that caused the later pandemics, the 1918 flu most likely began as a bird-adapted strain that, with just a handful of mutations, made itself at home in human beings. To flu researchers and public-health officials, the resemblance of the 1918 sequence to those of common avian flus underscores the stark fact that there is more than one way for a virulent strain like H5N1 to make the jump and become transmissible person to person. According to Taubenberger, this suggests a new strategy for surveillance, one that would include identifying and isolating a local variant of the virus on the verge of acquiring a complete complement of the essential mutations, after which point it would become impossible to contain.

What the genetic sequence of the 1918 virus did not reveal, however, was why the virus killed so ruthlessly, or how it made that critical leap to become transmissible. For those answers, they would have to take a more drastic step. "Jeff spent 10 years of his life doing this, and it told us nothing about pathogenicity," says Robert Webster, a noted flu researcher at St. Jude Children's Research Hospital in Memphis. "That's when we realized the sequence wasn't enough. It was necessary to put the damn thing together."

Necessary or not, the fact that it had become possible was probably enough to ensure that it would be done. In biology, the direction determined by what is possible has been downward, toward the exploration of ever more reduced levels of complexity. The progression started with the ancients, who first opened up the human body to ponder its organs and their functions. Once microscopes were developed in the 17th century, it became possible to observe the anatomy and behavior of the tissues and cells making up the organs, and with later advances, the proteins that build cells and determine their functions. In the last century we reached the level of the genes that conjure the proteins into being. Only in the last decade has automated sequencing made it possible to peer beneath genes at the individual letters of DNA constituting a complex organism's complete genome, including our own.

This is the bottom of the biological hierarchy, the fundament, where all of life rests upon the bedrock of inert information. Now that we have reached down this far, it becomes possible to use that information to do a U-turn and start back up, not just trying to understand life, but recreating and inventing it -- first simple viruses, but soon bacteria and other more complex organisms. The resurrection of the 1918 flu incarnates this turning point. It is not the first virus to be reconstituted from its genetic code. But it is so far the largest, and the meanest, and the only one to be snatched back into existence from a time when we knew so much less and were so much more at its mercy.

The wonder is not that scientists could reconstitute the "damn thing" from its genetic code. The wonder, and for some the fear, is that they could do it with so little effort or expense. Biosupply companies use synthesizing machines to build tiny pieces of DNA to order, using the sequence of letters in the virus's code. When placed in solution, these chemical snippets naturally assemble into longer pieces. With the help of a copying enzyme to fill in any gaps, the DNA molecules stitch themselves together into a complete gene, which can be inserted into a stable little circle of DNA called a plasmid -- packaged to go, so to speak. If you have plasmids containing all eight flu gene segments, it is a fairly simple matter to inject them along with some flu proteins into a cell and let nature take its course.

This method of building flu-virus particles from pure code is a clever application of the approach to understanding life called "reverse genetics" -- that is, looking at a gene to figure out its function, rather than the other way around. But it is not one requiring some spectacular insight or technological breakthrough. The method employs fairly routine molecular biology and was developed independently by two different flu teams, one at Mount Sinai School of Medicine in New York, the other at the University of Wisconsin. Peter Palese, from the Mount Sinai team, contacted Jeffery Taubenberger and suggested that if he would supply the blueprint for the virus, Mount Sinai would function as the parts factory, putting together the genes. Another laboratory, one with the biosecurity facilities required to work with highly infectious agents, would be recruited as the final assembly plant. That role would fall to Terrence Tumpey of the C.D.C.

The team did not even have to wait for Taubenberger to finish the whole sequence of the 1918 virus to begin testing its virulence. In 2001, Adolfo Garcia-Sastre and Christopher Basler, also at Mount Sinai, reconstructed the genes for just the two critical surface proteins and sent them on to Tumpey, at that time working at the Southeast Poultry Research Laboratory in Athens, Ga. Taking advantage of influenza's innate ability to mix and match genes from two strains, he combined the two 1918 genes with others from an innocuous laboratory strain to make a complete set. Tumpey infected some lab mice, which are normally not affected much by human flus. Five days later, he came into the laboratory at around 11 at night for a quick check on their progress. All the mice were dead.

In person, Tumpey is unnervingly imperturbable ask him what it's like handling an infectious agent that killed perhaps 50 million people, and he stares back at you and gives a little shrug. But this first demonstration of the virus's power got to him.

"I literally felt a chill go down my spine," he told me. "I knew I had this awesome virus, and Iɽ eventually be able to put the whole thing together."

He did not have much longer to wait. It took nearly 50 years to find a trace of the virus in preserved tissue, and nearly 10 years for Taubenberger to sequence its code, finishing the last of three genes driving the virus's replication machinery early last year. From that point, it required just a few months for the Mount Sinai group to transform the code into actual genes, and in Tumpey's lab mere days for the genes to begin assembling themselves into viable virus particles and come bursting out into the surrounding solution.

Tumpey and his colleagues were well aware that bringing such a lethal pathogen back into the world was going to cause controversy. But he was fairly certain that he had laid the groundwork to defend the decision, obtaining approvals from the highest levels at the C.D.C. and the National Institute of Allergy and Infectious Diseases, which had financed the work. He had conducted experiments showing that mice were protected from the virus by the current human flu vaccine and by Tamiflu, the antiviral drug. In any case, because a virus descended from the 1918 one has been circulating in the population since 1977, Tumpey is confident that everyone carries at least partial immunity to the 1918 virus itself.

Not everyone is as sanguine as Tumpey. "I believe that this was research that should not have been performed," says Richard Ebright, a Howard Hughes Medical Institute investigator at Rutgers University. "If this virus was to be accidentally or intentionally released, it is virtually certain that there would be greater lethality than from seasonal influenza, and quite possible that the threat of pandemic that is in the news daily would become a reality."

Neither Terrence Tumpey nor Richard Ebright really knows how vulnerable the population today would be to the resurrected virus. Nobody does. This uncertainty would seem to limit the virus's value as a bioweapon. In theory, anyone with nefarious intent and the requisite training in molecular biology could recreate the virus from the sequence published on the Internet. But why would any sensible bioterrorist go to such lengths to create a weapon that might do no more harm than a seasonal flu bug, or, if it did prove undiminished in its virulence, would kill his own people as indiscriminately as his enemies?

Then again, common sense is not a prerequisite for membership in a terrorist organization. Accidental release of the virus cannot be ruled out, either. While few question the experience and expertise of the C.D.C. in containing dangerous microbes, other labs will be working with the virus, and there is ample precedent for accidents occurring under stringent biosecurity, including release of the SARS virus in the past few years from three separate laboratories in Asia, which led to one death. In fact, the reason those of us who were not around in 1918 still may have some immunity to that pandemic strain is that a relatively innocuous descendant H1 type was reintroduced into the environment in 1977, probably by accident in China or Russia.

Given the potential danger, Robert Webster, the esteemed flu researcher who supported the reconstruction, is among those who say it would be better to conduct future research on the 1918 virus under Biosafety Level 4 conditions -- the maximum degree of security, used for working with lethal microorganisms like the Ebola virus and smallpox. But currently, only four institutions in the U.S. have functioning BSL-4 facilities, including the C.D.C. Imposing such restrictions would necessarily slow the progress of research.

This is something that Terrence Tumpey, among others, insists that we cannot afford. Earlier this month, the H5N1 virus recorded an extraordinary rash of cases, including four fatalities in Turkey, the first outside East Asia. All the victims appear to have caught the virus from eating or handling infected poultry. But most flu researchers worry that as the virus's range increases, so does the likelihood that somewhere, sometime, some random set of mutations will send it over the edge into transmissibility, unleashing a pandemic. Everyone agrees that at some point, another pandemic will come -- if not from this strain, then from some other one perhaps not even yet under surveillance. The best hope of containing its impact is to understand how it works. What are its mechanisms of infection and replication? How does it foil the host's immune response and jump from a conquered host to a fresh one?

In Tumpey's view, the 1918 virus is the star witness in a murder trial, and the interrogation should proceed without unnecessary impediments. Taubenberger's sequence can help indicate what questions to ask. Experiments with individual genes can suggest some possible answers. But only the living virus can reveal the full truth. The first round of interrogation is already under way. Using reverse genetics to test the contribution of any particular gene to the virus's pathogenicity, Tumpey and his colleagues can replace any target gene in the 1918 virus with its complement from a harmless strain, then measure the effect on the virus's potency. When he replaced the 1918 hemagglutinin gene with one from a garden-variety seasonal flu, the virus replicated at less than 1100th the rate in mice it was definitive proof of the essential role played by that gene in virulence. Tumpey already knew that the 1918 virus did not need one of the host's own enzymes to turn traitor and cleave apart the hemagglutinin protein to help the virus infect a cell. But when he created a 1918 virus without its own neuraminidase gene, this ability was lost, revealing that the virus toted its own cleaving mechanism into the host on that gene, like a butcher who brings his own knife. Meanwhile, Peter Palese's group has shown that another gene in the 1918 virus is especially good at blunting the human immune system's initial counterattack.

"It was perfect genes, working together, that made this virus what it was," Palese said. Then he gave a little laugh. "Or what it is."

Scientists can also examine the role in virulence and transmission of particular mutations on the virus's genes. Taubenberger's sequence again offers guidance. One of the large genes driving replication, for instance, bears a single mutation that is found not only in the 1918 virus, but also in all human flus. But no bird flus have this mutation -- not even H5N1. Is this mutation perhaps necessary for an avian virus to become transmissible from human to human? Combining reverse genetics with some other molecular tricks, you could insert that mutation into the gene of a nonvirulent avian flu, construct the virus and see how it behaves. The ultimate hope of such experiments is to uncover a clue to how the virus spreads or kills, and possibly a way to cripple it. Terrence Tumpey is already planning experiments with several research groups and companies that will use the 1918 virus to test possible antiviral drugs to block some universal mechanism of virulence, like the binding of hemagglutinin to the host cell. That work has added urgency, since the H5N1 flu appears to have developed resistance to one of two flu drugs currently on the market.

What may be the most informative research he intends to conduct must surely be the most dangerous as well. Tumpey's freezer contains the resurrected 1918 virus, which is lethal and highly transmissible. It also contains samples of the H5N1 virus, which is lethal but not yet transmissible. Using reverse genetics, he imagines "a great set of experiments" combining the genes of these two killers in various combinations, seeing if one might have the capacity to transmit from an infected animal model, like a ferret, to an uninfected one. This would create in the laboratory the very pandemic strain that researchers most fear may emerge at any time in nature. According to Tumpey, plans for these experiments are already "on paper." Needless to say, they will require complete approval first, and may have to be performed under Biosafety Level 4 conditions, since we would have no immunity to the recombinant organism.

For Richard Ebright, the prospect that the C.D.C. or some other lab would "jump the gun on nature" is worrisome under any circumstances. Other scientists and bioethicists are also calling for more independent, international review and control of further research on the 1918 virus and other synthetic pathogens yet to be concocted. It all comes down, of course, to whether what we can learn justifies the risk of bringing them into existence. While that debate moves forward, nature will go on conducting its own creative experiments, indifferent as always to our abilities to defend ourselves against them.

Jamie Shreeve is the author of "The Genome War: How Craig Venter Tried to Capture the Code of Life and Save the World." His last article for the magazine was about chimeras.