• Dengue viruses (DENV) is a mosquito borne viruses, belongs to the family Flaviviridae, the genus Flavivirus.
• Dengue viruses consist of five serotype named dengue virus types 1, 2, 3, 4 and 5 (DENV-1, -2, -3, -4 and -5), that are responsible for diseases.
• These serotypes are closely related but antigenically distinct.
• DENV-5 is a new serotype which has recently been described from Malaysia.
• Hotta and Kimura were the first to isolate the virus in 1943, by intracranial inoculation of serum from an acutely ill patient into suckling mice.

Morphology of Dengue virus:

• Dengue virus is spherical enveloped viruses with single-protein capsids, two membrane proteins, envelope and membrane.
• It has a single-stranded Positive-sense RNA genome of ~10,700 nucleotides, surrounded by a nucleocapsid and covered by a lipid envelope that contains the viral glycoproteins.
• The RNA genome lacks poly A tail at 3’end and contains a single open reading frame (ORF) flanked by two untranslated regions (5’ and 3’UTRs).
• The 5’ and 3’ terminal RNA sequences of the genome form large stem loop structures known as stem loop A (SLA) and 3’ stem loop (3’ SL) respectively, both essential for viral replication.
• The single ORF encodes a precursor polyprotein, which is co- and post-translational cleavage resulting in the formation of three structural proteins, Capsid (C), membrane (M), and envelope (E), and seven non-structural proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5. NS2B and NS3 form the proteases, and NS3 also has helicase and RNA triphosphate activity; NS5 is the RNA dependent RNA polymerase and also has methyltransferase activity.
• It has four serotypes, DEN-1, DEN- 2, DEN-3 and DEN-4, which are capable of causing dengue fever (DF).

Pathology and clinical features of Dengue:

• Dengue is an acute infectious vector borne disease caused by dengue viruses and transmitted to human by the Aedes species, primarily by Aedes aegypti.
• The prevalence of dengue viral infection has been estimated that over 2.5 billion people live in the areas of risk with an incidence of 50-100 million cases per year and several thousands of deaths are estimated to occur annually worldwide, resulting in 500,000 cases of DHF/DSS and 25,000 deaths.
• Dengue virus induces clinical illness ranging from asymptomatic or mild febrile illness, i.e., Dengue Fever (DF) to severe disease forms, i.e., Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).
• Infection with any one of the four DENV serotypes has the potential to involve all human organ systems and cause a wide variety of clinical manifestations ranging from mild febrile illness to severe and fatal disease.
• Symptomatic dengue disease is separated into two different clinical syndromes, dengue fever (DF), and the more severe dengue hemorrhagic fever (DHF); DF was described as a nonspecific febrile illness with prominent constitutional symptoms, while DHF was defined as a distinct syndrome characterized by increased vascular permeability, altered hemostasis and hemorrhage. DF is also referred to as the ‘break bone’ disease due to its nature of severe joint and muscular pain.
• Following an infectious mosquito bite there is an incubation period of up to 2 weeks (commonly 5-7 days), after which the individual develops symptoms suddenly and the illness typically follows three phases- an initial febrile phase, a critical phase starts 4-5 days from fever onset, followed by a spontaneous recovery phase.
  • In febrile phase, patient experiences sudden onset of high fever (39-40°c) accompanied by nonspecific constitutional symptoms including headache, general malaise, nausea, vomiting, myalgia, and joint pain.
  • In recovery phase, the increased vascular permeability and abnormal hemostasis are transient and usually resolve within 48-72 hours. Spontaneous reabsorption of fluid starts around 6-8 day of illness and progress rapidly, usually concurrent with improvement in the patient’s symptoms. Loss of hair has been reported during convalescence.

Primary infection:

• Primary infection with dengue virus results in a self-limiting disease characterized by mild to high fever lasting 3 to 7 days, severe headache with pain behind the eyes, muscle and joint pain, and a rash.
• Complications often occur within two days after the fall in temperature. Epistaxis, bleeding of gums, passage of black stools, rashes (petechiae, maculopapular, bruises, etc.) and sub-conjunctival hemorrhage are indicators of increasing disease severity.
• A maculopapular rash usually appears 3 – 4 days after the onset of fever. Secondary infection with a different dengue virus serotype is the more common form of the disease in many parts of Southeast Asia and South America.
• The major clinical symptoms can include high fever, hemorrhagic events, and circulatory failure, and the fatality rate can be as high as 30%. Early diagnosis of dengue shock syndrome is particularly important, as patients may die within 12 to 24 hr if appropriate treatment is not administered.

The major host factors that influence clinical manifestations are:
• Age: constitutional symptoms become more prominent with increasing age, that adults complain of headache, retro-orbital pain, and severe myalgia and arthralgia more frequently than children.
• Gender: Females have a lower threshold for vascular leakage than males. Although dengue is diagnosed more frequently in male than female patients, female patients have a higher risk of developing DSS and of dying from this complication than male patients [34].
• Pregnancy and transplacental infection: With the increasing burden of dengue seen among young adults, exposure to infection during pregnancy is becoming more frequent.

• Clinical findings alone are not sufficient to make an accurate diagnosis of DENV as several other infectious diseases may present with similar findings, which requires the need for laboratory testing for the dengue confirmation.
• Leucopenia, thrombocytopenia, increased hematocrit and liver enzyme levels are common laboratory findings suggestive of DENV infection.
• Serological tests, such as rapid diagnostic test (RDT), Enzyme-linked immunosorbent assay (ELISA) are widely used.
• In Nepal, serological testing has been used as an important tool for diagnosis of suspected DENV infection during outbreaks.

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Pathogenesis of Dengue:

• Dengue virus after entering in the body invades the local macrophages and multiply there.
• Infected local cells then migrate from site of infection to lymph nodes, where monocytes and macrophages are recruited, which become targets of infection.
• Consequently, infection is amplified and virus is disseminated through the lymphatic system. As a result of this primary viremia, several cells of the mononuclear lineage, including blood-derived monocytes
• Viremia develops within 24 hours. During this period, virus travels throughout the body.
• Bone marrow cells have also been shown to be susceptible to infection with DENV
• In severe case, viral load is very high and many vital organs are affected.
• Virus infected macrophages produces a number of signaling proteins such as interferons, cytokines, chemokines, TNF, other mediators  which are responsible for many symptoms such as flue like syndrome and pain.
• These mediators affects hemostatic system of body.
• Fluid from blood vessels starts to leak out so that the blood volume decreases resulting in low blood pressure.
• Decrease in blood pressure causes insufficient supply of blood and Oxygen to vital organs such as brains.
• Dengue also infects bone marrow, so that bone marrow cannot produces sufficient platelets.
• Since platelets are needed for blood clotting, dengue infection causes blood clotting defect and increase the risk of bleeding.

Primary infection:

• Primary infection is characterized by fever, break bone fever, retro-orbital pain and flue like syndrome.
• The person who is not previously infected with any Flavivirus is termed as primary infection.
• In primary infection ratio of Dengue specific IgM to IgG is high.

Secondary infection:

• Dengue infection in host who is immunologically sensitized to dengue or other flavivirus is termed as secondary infection.
• Secondary infection is characterized by rise in antibody titer. The ratio of IgM to IgG is low.

Clinical manifestation:

• Dengue fever is symptomatic or asymptomatic.
• Clinical disease onsets 4-6 days after infective mosquito bite.

Symptomatic dengue includes, classical dengue fever, Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS).

I. Classical dengue fever:

• It is characterized by fever, rashes, severe headache, pain behind eyes, pain in muscle and joints, enlarged lymph nodes,
• Fever lasts for 2-7 days
• Myalgia and break bone fever ( deep bone pain) is the characteristic of Dengue fever
• Classical dengue fever is self-limited.
• Mostly adults and older children are affected,

II. Dengue hemorrhagic fever (DHF):

• DHF is marked by bleeding from skin and mucus membrane.
• DHF has four major clinical manifestation- High fever, hemorrhagic phenomenon, hepatomegaly and circulatory failure.
• In early stage of infection DHF resembles classical dengue fever.
• Usually occurs in children
• About 23% of children with DHF develops circulatory failure with haemo-concentration and a marked decrease in platelets counts leading to severe hemorrhage.

III. Dengue shock syndrome (DSS):

• DSS is serious form of DHF.
• All serotypes of Dengue are associated with DHF and DSS.
• The cause of DSS is not clearly understood. But it is assumed due to antibody dependent enhancement (ADE).
• Pre-existing heterologous antibody against particular dengue serotype binds with the new infected virus serotype by its Fab region and Fc region of antibody binds to receptor on macrophage. So this pre-existing antibody increases virus infection to healthy macrophages.
• From recent outbreak information of DHF, patients infected with DEN-2 after primarily infected with other serotypes can leads to DSS.
• DSS is characterized by circulatory failure, rapid pulse, abnormal pain, severe bleeding from GI tract and other organs.
• Usually occurs in children

Lab diagnosis:

Sample: blood, serum

1. HIA (Hemagglutinin Inhibition assay): test antibody against hemagglutinin spike of virus.

2. FAT (Fluorescence antibody test): detects virus antigen using virus specific antibody

3. MAC-ELISA (IgM capture ELISA): detects Dengue specific IgM antibody.it can distinguish primary infection from secondary infection. In primary infection ration of IgM to IgG is greater than 1.5

4. Neutralization test: detect antibody against dengue

5. Virus isolation: mosquito cell line culture with patient serum.

6. Nucleic acid detection: RT-PCR is used to detect virus genome.

Treatment:

• No specific treatment for dengue.
• Early and proper diagnosis can reduce symptoms and prevents complication and death
• Vaccine:
• Dengvaxia vaccine is licensed and available in some countries for people ages 9-45 years old. The World Health Organization recommends that the vaccine only be given to persons with confirmed prior dengue virus infection.
• The vaccine manufacturer, Sanofi Pasteur, announced in 2017 that people who receive the vaccine and have not been previously infected with a dengue virus may be at risk of developing severe dengue if they get dengue after being vaccinated.

Dengue pathogenesis, clinical manifestation, lab diagnosis and treatment

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Introduction

• Dengue is an arthropod borne virus belongs to family Flaviviridae and genus Flavivirus.
• Dengue virus is transmitted between people by female mosquitoes Ades aegypti and Ades albopidus, which are found throughout the tropical and sub-tropical regions.

Structure of Dengue virus:

• Dengue virus is spherical enveloped viruses with approximately 40-60 nm in diameter.
• Dengue virus has single-stranded Positive-sense RNA genome of about 11 Kbp long, surrounded by a nucleocapsid and covered by a lipid envelope that contains the viral glycoproteins.

I. Genome:

• Dengue has + SS RNA as genome which contains 3 structural gene and 7 non-structural gene.
• The RNA genome lack poly A tail at 3’end
• Genome contains a single open reading frame (ORF) flanked by two untranslated regions (5’ and 3’UTRs).
• The 5’ and 3’ terminal RNA sequences of the genome form large stem loop structures known as stem loop A (SLA) and 3’ stem loop (3’ SL) respectively, both essential for viral replication.
• Structural gene codes for core protein (capsid), membrane associated protein and envelope protein.
• Non-structural gene codes for non-structural proteins such as NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 which helps in viral replication.
• NS5 is the RNA dependent RNA polymerase.

II. Capsid:

• Icosahedral capsid, 20-30 nm in diameter
• Spherical shape

III. Envelope:

• Envelope contains glycoprotein spike which has haemagglutinin activity.

Serotypes of Dengue

• There are 4 serotype of Dengue designated as DEN-1, DEN-2, DEN-3 and DEN-4
• All 4 serotypes have +ss RNA as genome of 11 Kbp length.
• DENV-5 is a new serotype which has recently been described from Malaysia

Mode of transmission:

• Dengue is transmitted by female Ades aegypti and Ades albopidus
• In order to transmit dengue between person, Mosquitoes must feed blood of patient during 5 days period when large amount of virus are present in blood.
• Inside mosquito, virus requires additional 8-12 days incubation period before it can be transmitted to healthy person.
• The dengue virus mixed with saliva of mosquito and transmitted intradermally during biting.
• In rare cases dengue is transmitted through blood transfusion, organ transplant and from infected mother to foetus.

Dengue virus: structure, serotypes and mode of transmission

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