dengue virus

Indian scientists break dengue code, develop non-infectious vaccine from yeast.

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Indian scientists have achieved an important breakthrough in their efforts to develop a vaccine to prevent the deadly dengue. Supported by the Department of Biotechnology under the Ministry of Science & Technology, scientists at International Centre for Genetic Engineering and Biotechnology (ICGEB) in New Delhi have developed a non-infectious dengue vaccine from yeast.

Preliminary animal trials of the vaccine have yielded good results.

“Search for a dengue vaccine has been going on across the world for past several decades. We, at our centre, started experiments seven years ago. The new technology we have used, i.e. recombinant DNA technology, to develop the dengue vaccine is a breakthrough,” said Dr Navin Khanna, group leader of Recombinant Gene Products Group, ICGEB. The initial trials done on mice gave encouraging results.

The research team explored virus-like particles which can provide “robust immunity” against the vector-borne disease that is endemic to more than a hundred countries. “There are four closely related dengue viruses (DENVs) that cause this disease. A vaccine that can protect against all four DENVs is an unmet public health need,” said Dr Khanna.

Explaining the need to explore a new technology to develop the vaccine, he said: “Efforts to develop a live attenuated vaccine (a vaccine created by reducing the virulence of a pathogen but still keeping it viable) have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This underscored the need to experiment with non-replicating vaccine options.”

Among the disadvantages of the vaccine developed by live attenuated technology is that it can cause severe complications in patients with low immunity.

The ICGEB scientists used the yeast ‘Pichia pastoris‘ to develop dengue virus-like particles. “Using recombinant DNA technology, we have created non-infectious dengue virus-like particles made of only the major DENV ‘envelope protein’ important for eliciting virus-specific immunity.

These virus-like particles elicit high levels of virus-neutralising antibodies which protected the mice significantly against lethal DENV challenge,” said Dr Khanna. “The encouraging data obtained for virus-like particles specific to one of the four DENVs warrant the development of virus-like particles specific to the remaining three DENV strains,” he added.

Neurological complications of dengue virus infection.

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Dengue is the second most common mosquito-borne disease affecting human beings. In 2009, WHO endorsed new guidelines that, for the first time, consider neurological manifestations in the clinical case classification for severe dengue. Dengue can manifest with a wide range of neurological features, which have been noted—depending on the clinical setting—in 0·5—21% of patients with dengue admitted to hospital. Furthermore, dengue was identified in 4—47% of admissions with encephalitis-like illness in endemic areas. Neurological complications can be categorised into dengue encephalopathy (eg, caused by hepatic failure or metabolic disorders), encephalitis (caused by direct virus invasion), neuromuscular complications (eg, Guillain-Barré syndrome or transient muscle dysfunctions), and neuro-ophthalmic involvement. However, overlap of these categories is possible. In endemic countries and after travel to these regions, dengue should be considered in patients presenting with fever and acute neurological manifestations.

Diagnosis

Clinical suspicion is essential for diagnosis of dengue because many symptoms are non-specific. Various methods are available for laboratory confirmation. During the first days of infection, dengue virus is present in blood; thus, at that time, detection of NS1 antigen or RNA by RT-PCR and viral culture are appropriate diagnostic methods.1 Dengue virus-specific IgM antibodies are present in serum samples 3—10 days after disease onset.1 IgM capture (MAC)-ELISA is the most widely used serological test. Antibodies against other flaviviruses (eg, Japanese encephalitis, West Nile virus, yellow fever) might cross-react with dengue virus, leading to false-positive reactions.136

In endemic countries, or among travellers who recently (<14 days) returned from such regions, dengue should be ruled out in patients with fever and neurological features (panel 2). If possible, lumbar puncture should be done and CSF analysed for abnormalities and for dengue virus-specific antibodies, NS1 antigen, or dengue virus RNA, depending on available laboratory facilities. Differential diagnosis in patients with febrile encephalopathy includes malaria, tuberculosis, leptospirosis, rickettsial infection, and other bacterial or viral diseases (caused by, for example, Japanese encephalitis, West Nile virus, or herpes simplex virus [HSV]), depending on the local epidemiology. In a prospective hospital-based study in Vietnam, most children with acute encephalitis of presumed viral origin were infected with Japanese encephalitis (26%), followed by enteroviruses (9%) and dengue virus (5%).30 In adults and adolescents in Brazil, dengue was the leading cause of viral encephalitis (47%), followed by infections with HSV-1.31

To differentiate dengue encephalitis from encephalopathy, detection of dengue virus, NS1 antigen, or dengue virus-specific IgM antibodies in CSF is helpful. Nevertheless, sensitivity of serological techniques can be low. Dengue virus-specific IgM antibodies have been recorded in CSF of 22—33% of patients diagnosed with dengue encephalitis (Table 1, Table 2).90Detection of dengue virus in CSF could be hampered by low sensitivity of RT-PCR in CSF, compared with findings in serum, because of a lower viral load.137 Moreover, measurement of IgM antibodies in CSF might not be a reliable diagnostic marker of dengue CNS involvement, owing to low titres in CSF.138 Abnormalities in CSF—such as lymphocytic pleocytosis—support the diagnosis of dengue encephalitis, but they are not always present (Table 1, Table 2, Table 3). A mild increase in CSF protein has been recorded.28 In a series of patients with neurological complications of dengue, four of seven with encephalitis had no alterations in CSF.90 Therefore, normal CSF cellularity should not exclude dengue encephalitis.

The case definitions in panel 2 are designed to be used epidemiologically and clinically and to guide diagnosis and prognosis. Although we propose criteria for a classification scheme, a topic as challenging and as controversial as dengue encephalitis needs to be addressed in a standard way. Prospective studies are needed to assess the specificity and sensitivity of the proposed case definitions and to generate supporting evidence. Cases fulfilling neither the definition for encephalitis nor that for encephalopathy—eg, without CSF testing or when categories are overlapping—can be categorised as other or non-specified dengue CNS involvement.

Neuroimaging might provide additional clues in the diagnosis of neurological complications of dengue. In dengue encephalitis, brain MRI can be normal or show focal parenchymal abnormalities.22, 41 Nevertheless, no specific MRI findings suggestive of dengue encephalitis have been reported. Neuroimaging features of patients with dengue are diverse, with cerebral oedema the most commonly reported finding.77 Meningeal enhancement on post-contrast MRI has been reported occasionally as well.77

Finally, EEG abnormalities can be seen in dengue patients with neurological complications. In a study of 23 patients with dengue virus infection and neurological symptoms, EEG abnormalities were recorded in 12 people.139 Slowing on EEG can be seen, but this finding is unspecific and could be attributable to seizures, intracranial haemorrhage, and viral infection per se, besides encephalopathy.77

Management

Currently, no effective antiviral agents are available to treat symptomatic dengue virus infection.140 Therefore, management remains supportive. In mild cases, antipyretic drugs and oral fluids could be useful. Acetyl-salicylic derivatives and other non-steroidal anti-inflammatory drugs should be avoided. Management of haemorrhagic complications should be initially conservative. Precise management of intravenous fluids is needed, and blood or platelet transfusion is only necessary when severe bleeding takes place.1

In patients with severe dengue and signs of plasma leakage, prompt fluid resuscitation is imperative, with close monitoring of packed-cell volume to avoid fluid overload. Isotonic crystalloid solutions should be used, with isotonic colloid solutions reserved for patients presenting with profound shock or those who do not have a response to initial crystalloid treatment.140, 141 In a randomised controlled trial from Vietnam,142 use of oral prednisolone during the early acute phase of dengue infection was not associated with a reduction in the development of shock or other recognised complications of dengue virus infection.

For supportive management of patients with neurological manifestations, possible underlying causes such as intracranial bleeding, liver failure, hyponatraemia, hypokalaemia, or metabolic acidosis should be ruled out and—if possible—corrected. Management of dengue encephalitis remains supportive and should include adequate hydration, nutrition, monitoring of consciousness, and maintenance of airways.143 Symptomatic seizures should be treated with non-hepatotoxic anticonvulsants. Decompressive craniotomy and cerebral haematoma evacuation were done in two patients with dengue after correction of prothrombin time and platelet count.92 Nevertheless, prognosis is not good and, in one case series, two of five patients died.92 At this moment, haematoma surgery cannot be proposed as a routine treatment for dengue virus intracranial bleeding.

Some clinicians recommend treatment of immune-mediated dengue CNS involvement with pulses of intravenous methylprednisolone for several days.50, 69, 72 However, up to now, no randomised controlled trial has been undertaken to show the efficacy of this approach in patients with dengue myelitis or acute disseminated encephalomyelitis. High doses of intravenous immunoglobulin might be useful to treat post-dengue Guillain-Barré syndrome. Supportive treatment—including hydration and analgesic drugs—is used for myalgia and transitory muscle dysfunction. The effectiveness of corticosteroids in dengue myositis remains to be proven.

No treatment has been approved for neuro-ophthalmic manifestations of dengue. Steroids have been administered previously because of possible underlying immune mechanisms, although up to now no randomised trials have been done. Topical steroids have been used to treat anterior uveitis, whereas pulsed intravenous methylprednisolone or systemic oral steroids might be indicated for extensive retinal vasculitis.120

Currently, no vaccine is available for protection against dengue. However, several vaccine candidates are in development.

Conclusions and future research

Dengue should be included in the differential diagnosis of acute febrile disease with neurological manifestations in dengue-endemic countries and in patients with a recent travel history to an endemic region. Many neurological manifestations of dengue have been recorded, ranging—with substantial overlap—from encephalitis and encephalopathy to immune-mediated syndromes and muscle involvement. Recent evidence suggests that dengue virus has neuroinvasive capacity. In several studies in endemic areas, a large proportion of viral encephalitis was caused by dengue virus.26—32 However, even though CNS involvement is included now as a criterion for severe dengue in the 2009 WHO case classification,1 no standardised case definitions or diagnostic criteria for dengue encephalitis or encephalopathy have been agreed, which leads to inconsistent use of these terms in published work.

An updated WHO dengue guideline should include a case definition for dengue encephalitis and encephalopathy, to guide clinicians and clinical epidemiological researchers into this topic. A case classification—such as the one proposed in panel 2—could serve as a starting point, which could be reviewed by WHO, agreed by consensus and best available current evidence, and refined as additional data become available from prospective studies. For this reason, assessment of CSF in patients with suspected neurological manifestations of dengue should be standardised. Very few published reports present findings of CSF testing for dengue virus, dengue virus-specific IgM antibodies, or NS1 antigen combined with CSF cellularity and confirmation of dengue in serum samples in a consistent way. Further epidemiological and neuropathological studies are needed to ascertain the true incidence and burden of neurological complications of dengue, to elucidate the underlying pathophysiology, and to assess the sensitivity and specificity of diagnostic markers for dengue encephalitis.

Source: Lancet

Possible new route to fight dengue virus pointed.

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Researchers have identified enzymes and biochemical compounds called lipids that are targeted and modified by the dengue virus during infection, suggesting a potential new approach to control the aggressive mosquito-borne pathogen.

Findings also suggest that medications used to treat high cholesterol and other lipid-related conditions might also inhibit dengue’s replication and could represent a potential new therapy. The researchers have identified how infected mosquito cells undergo changes to certain lipids in membranes and in biochemical sensors that alert cells of invading viruses.

“The virus reorganizes the internal architecture of the cell to support its own needs,” said Purdue University research scientist Rushika Perera. “Many details are unknown. This is our first attempt to understand how the virus alters lipids as part of the infection process. Part of what we looked at in this work was how the virus changes the cell, and the next step will be to figure out why.”

 

The researchers uncovered new details of how the virus alters lipids in membranes surrounding structures inside cells called organelles, including the mitochondria, which provide energy critical for a cell to function, and theendoplasmic reticulum, where proteins and lipids are synthesized.

“Findings also show that important host enzymes are used by the virus and may be targets for future antiviral drugs,” said Richard J. Kuhn, a professor and head of Purdue’s Department of Biological Sciences and director of the Bindley Bioscience Center. “It turns out, the pills you take to control your cholesterol might have some capability to control dengue.”

The work was led by Perera in collaboration with researchers at Purdue’s Bindley Bioscience Center and the Pacific Northwest National Laboratory. Findings are detailed in a research paper to appear Thursday (March 22) in the journal PLoS Pathogens.

The findings could apply to viruses similar to dengue, including the West Nile virus, yellow fever and hepatitis C.

Dengue causes 50 million to 100 million infections per year and is considered one of the most aggressive mosquito-borne human pathogens worldwide. It is a leading cause of serious illness and death among children in some Asian and Latin American countries.

“Identifying pathways of infection will help us understand how these viruses work,” Kuhn said. “Many viruses, including dengue, dramatically alter a host cell upon infection, and in this paper we begin to dissect the precise changes that occur. Ultimately, we are trying to understand how the virus subverts and exploits the host and uses it for its own purpose, which is to replicate.”

The team learned specifically that an enzyme called fatty acid synthase, which cells use to synthesize lipids, is affected by the virus. Researchers showed that compounds inhibiting production of the enzyme also inhibit virus replication, suggesting drugs already on the market to treat diseases related to lipid synthesis and storage, including diabetes and cancer, also might be used to treat dengue, Kuhn said.

 

The research paper was written by Perera; Kuhn; Bindley researchers Catherine Riley, Amber S. Hopf-Jannasch and Jiri Adamec; and PNNL researchers Giorgis Isaac, Ronald J. Moore, Karl W. Weitz, Ljiljana Pasa-Tolic and Thomas O. Metz.

The researchers had previously studied a compound that inhibits the production of fatty acid synthase in human cells. In the new findings, the researchers showed that the virus commandeers some of the same enzymes in both mosquito and human cells, meaning the same compound could work to attack the virus in mosquito cells.

“This is important because it may be easier to control the virus in mosquitoes than in humans,” Kuhn said.

Globally, dengue has grown dramatically in recent decades, placing about half the world’s population at risk of infection. The infection causes flulike illness and occasionally develops into a potentially lethal complication called dengue hemorrhagic fever. Prompt medical care for this severe form of dengue virus infection has been shown to decrease mortality rates from more than 20 percent to less than 1 percent, according to the World Health Organization.

 

The research hinges on recent advances in two areas: high-resolution mass spectrometry and “informatics,” or using computers to process volumes of information.

“You generate a large quantity of data that has to be interpreted in terms of what molecules you are looking at,” Perera said. “The mass spectrometer takes hundreds of lipids and breaks them apart, and then a computer is needed to put it all back together and identify them. It’s only in the past five years or so that we’ve had the capability to do this with the required accuracy.”

The researchers also detail changes in the curvature of membranes, using another technique called cryoelectron microscopy, and pinpointed an isolated part of the cell where most of the virus replication takes place, a complex of membranes modified by the infection. The virus is thought to commandeer enzymes, relocating them to this region where virus replication factories are situated.

Because the research tools enable scientists to see how changes in membranes and signaling lipids alter how a cell functions, a long-term benefit of the research is learning how to use a virus as a tool to better understand cellular processes, Perera said.

Dengue virus causes ~50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

Source: Purdue University