Dengue: a research perspective Speaker: Alan L. Rothman, MD Center for Infectious Disease and Vaccine Research University of Massachusetts Medical School audio presentation

Highlights

	There are four dengue viruses, referred to as serotypes.
	Immunity to the same serotype is long lasting; immunity to other serotypes is partial and transient.
	More serious symptoms tend to be seen in secondary, rather than primary, infection.
	Plasma leakage distinguishes dengue fever from dengue hemorrhagic fever.
	Both viral and host factors influence the risk of developing dengue hemorrhagic fever.

Dengue virus infection: complex, multifaceted

Dengue is actually four viruses, not one. Because the viruses are defined based on serologic responses, they are referred to as dengue serotypes. "In understanding the implication of this, it has to be borne in mind that at least to the best of our knowledge, immunity to the same serotype-homotypic immunity—is long lasting, perhaps life long. But infection with one serotype confers, at best, only partial and transient immunity to other serotypes," explained Alan L. Rothman of the Center for Infectious Disease and Vaccine Research at the University of Massachusetts Medical School. "After this point, people who've been exposed to one virus are now susceptible again to infection with any of the other three serotypes of dengue virus. So we refer to the first time people are exposed to dengue as primary infection and subsequent exposure as secondary infection."

Dengue is transmitted to humans by mosquitoes, primarily Aedes aegypti. Most transmission occurs in urban areas, where infection can be endemic or epidemic. Humans and other primates seem to be the only natural vertebrate hosts for dengue virus infection; however, the viral strains that infect both groups are genetically distinct.

The risk of serious symptoms is greater in secondary infection.

Infection starts when the virus is injected, and viral replication begins at the inoculation site. Within about a day, however, virus can be found in regional lymph nodes. "Presumably, further replication goes on in the lymph nodes, and at some point the viral load increases to the point where it becomes spread throughout the body and disseminated. At this point, we detect viremia," Rothman continued. Within 12 to 24 hours, symptoms—for example, fever, malaise, headache, fatigue—appear. When the fever disappears, viremia ends and viral load declines rapidly.

The risk of more serious symptoms—bleeding diathesis and changes in vascular permeability, for example—seems to be greater in secondary, rather than primary, infection. This and other factors suggest an involvement of the immune response in the pathogenesis of dengue hemorrhagic fever, a life-threatening form of the disease.

From infection to disease

Many researchers are attempting to elucidate the connections between virus infection and disease, and to determine who is at greatest risk of developing dengue hemorrhagic fever. However, the models currently used to study the virus all have drawbacks, Rothman noted. For example, findings from cell culture systems for studying viral entry, replication, protein-protein interactions, and cellular responses to infection may be influenced by the type of viral strains used—that is, whether they are lab-adapted or natural.

Animal models also present problems. Primates, which are natural hosts for dengue virus, do not develop disease. So although primate studies can be used to study viral replication, they yield no information on what's happening in humans. Mice studies yield equivocal results, depending on the strain, and again, no disease that is comparable to human dengue fever.

Clinical studies also have drawbacks. Experimental challenges "pose risks [to participants] that are difficult to define," said Rothman. Studies of natural infections are subject to, among other factors, population differences (e.g., genetics, viral strains, environmental factors), lack of knowledge of previous exposures and immunologic history, a low virus isolation rate in the late phases of illness, and imprecise definitions of disease.

Despite the lack of a good disease model, researchers are making significant headway in understanding dengue virology immunology, and pathogenesis, said Rothman, who presented a detailed overview of current knowledge in these areas. Some key findings of dengue virology include: elucidation of the structure of the E glycoprotein, the major surface glycoprotein of dengue fever; the recent observation that dendritic cells are highly susceptible to dengue in vitro and in vivo, and that DC-SIGN is a key viral receptor; and the notion that dengue virus inhibits interferon signaling, and that this is a key component of the interaction of dengue with the host cell.

Recent immunologic studies have shown that the major targets of the antibody response to dengue are the E glycoprotein and a non-structural protein, NS1. Smaller proteins such as preM protein and the non-structural protein NS3 are also involved, but in ways that are not yet understood.

Antibodies to the preM protein, E protein, and NS1 are cross-reactive across viral serotypes, suggesting that these proteins could be participating in secondary infection, Rothman noted. Antibodies to the E protein may also play a role in disease pathogenesis via enhancement of infection—the hypothesis that antibodies present from a previous dengue infection can bind to the virus but are not able to neutralize it, and may actually increase viral uptake into cells such as monocytes that have immunoglobulin receptors.

CD4 and CD8 responses are also cross-reactive with other serotypes. Thus, in a single individual, said Rothman, "you have some determinants in which the T-cell response links only to the same serotype, and other determinants that include T cells that react with either one or all of the other dengue serotypes." In the examples he showed, several individuals had robust responses to the virus with which they were infected, but also cross-reactive responses to at least one of the other serotypes.

Moreover, "cross-reactivity is not an all or none phenomenon," he explained. "We've observed what we call a partial agonist effect, in which T cells may recognize a particular peptide that differentiates, for example, dengue 2 from dengue 3, but the cells are not able to induce an interferon gamma response or proliferate fully. So again, this shows a great deal of complexity in the response."

Who's at risk for dengue hemorrhagic fever?

Rothman devoted the final section of his talk to reviewing the viral and host factors that seem to increase the risk of developing dengue hemorrhagic fever. He began by emphasizing that fever, headache, and myalgias are all common symptoms of both dengue fever and dengue hemorrhagic fever—they don't distinguish them.

Plasma leakage distinguishes dengue fever from dengue hemorrhagic fever.

"Rash can be present or absent in both dengue fever and dengue hemorrhagic fever," he noted. "Thrombocytopenia is used in the definition of hemorrhagic fever, and so it's almost always severe in these patients; however, some patients who have dengue fever also have severe thrombocytopenia, so this feature alone can't be used to define groups. Bleeding can be present or absent even in dengue hemorrhagic fever, and that's been very difficult for groups new to dengue to take into consideration. Plasma leakage, by contrast, is seen only in dengue hemorrhagic fever and so that is the criterion."

Many research groups have identified different viral and host factors that affect the risk of hemorrhagic fever. One potentially important viral factor is serotype. In a study of Thai children by Vaughn and colleagues, for example, about one-third of those symptomatic with dengue serotype 1 or 3 infection had primary infection whereas symptomatic primary infection with serotypes 2 or 4 was rarely seen.

The genotype of the virus also seems to play a role. Rothman reviewed molecular epidemiologic data on two distinct genotypes of dengue 2 virus: a Southeast Asian genotype and an American genotype. The Asian genotype that has been circulating in Asia since the 1950s was introduced into the Americas in the 1980s, and has been associated with epidemic dengue hemorrhagic fever in many regions. "And yet we now have convincing data that another group of viruses that had been circulating in the Americas in the 1960s through the 1980s, and in Peru in the 1990s, do not seem to be associated with hemorrhagic fever," he said. "They just don't seem to be capable, even when causing secondary infection, of causing the hemorrhagic fever syndrome." Differences in the non-coding regions of the gene and a single amino acid substitution in the E protein may relate to the ability of the Southeast Asian genotype, but not the American genotype, to replicate in dendritic cells, noted Rothman, "and so understanding this is going to be a key component of understanding disease pathogenesis."

Host factors that affect disease severity include genetic associations (for example, a study by Stephens and colleagues of HLA association showed that HLA-A*0207 was overrepresented in patients with severe disease, whereas -A*0203 was overrepresented in patients with mild disease); antibody and T-cell responses, particularly in the presence of previous infection; cytokine production; and levels of viremia.

The bottom line is that there is an interaction among both viral and host factors.

The bottom line is that there is an interaction among both viral and host factors, concluded Rothman. "When viremia is low, the antigen burden is low, and regardless of the T-cell background, cytokine production will be low and the risk of dengue hemorrhagic fever will be low." By contrast, he explained, "when you have high viremia and high antigen burden, we speculate that the outcome will relate to the T-cell profile, and it's only when you get a high TNF response that your risk of dengue hemorrhagic fever is elevated."