1. Dr. Sadia Anjum
Lecture 7,8

2. Dengue Viruses
The dengue viruses are members of the genus Flavivirus in the family Flaviviridae.
Along with the dengue virus, this genus also includes a number of other viruses transmitted by mosquitoes and ticks that are responsible for human diseases. 
Flavivirus includes the yellow fever, West Nile, Japanese encephalitis, and tick-borne encephalitis viruses.

3. Dengue Virus
There are four different strains of dengue virus. DEN-1, DEN-2, DEN-3, and DEN-4. These four viruses are called serotypes because each has different interactions with the antibodies in human blood serum. 
These serotypes are very similar (65%) so similar in fact that the immune system recognizes all of them after seeing only one. 
 In the first infection virus particles will be captured and processed by so-called antigen presenting cells. 
These viruses will be presented to T-cells causing them to become activated. And likewise B-cells will encounter their antigen free floating and become activated. B-cells produce antibodies. 
Human T-cells each are programmed to recognize a specific pattern (or antigen). Antibodies are used (among other things) to tag the viruses to encourage their uptake by macrophages (called opsonization) and inactivate them.

4. The change in distribution of dengue serotypes
The distribution of dengue serotypes in 1970 (a) and 2004 (b). Guzman, M. G. et al. Dengue: A continuing global threat. Nature Reviews Microbiology 8, S7–S16 (2010). All rights reserved

5. Genome and Structure
The dengue virus genome is a single strand of RNA. It is referred to as positive-sense RNA because it can be directly translated into proteins.
The viral genome encodes ten genes. The genome is translated as a single, long polypeptide and then cut into ten proteins.

6. Den V proteins and their functions

7. Den V proteins and their functions
NS1, associates with the membrane on the cell surface and in the RNA replication complex.
NS1 lacks a transmembrane domain, and it associates with the membrane  via lipid rafts.
Smaller M protein Apart from fprming the prM–E complex, little is known about the cellular or biochemical properties of prM/M.
M is capable of inducing apoptosis in a sequence and localisation-dependent manner , and  and interacts with host proteins during the entry and assembly stage of the virus lifecycle
It act as chapron for folding of E protein

8. Virus Replication and release

9. Viral binding and infection
Dengue virus binds to its receptor, and this process is mediated by envelop protein (E).
In mammalian cell, DEN 1–4 serotypes bind with Heparan sulfate, nLc4Cer, DC-SIGN/L-SIGN and Mannose receptors.
DEN-2 serotype also binds with HSP70/HSP90, GRP78, CD14- associated protein and two unknown proteins having trypsin resistance and trypsin sensitive properties.
DEN 1–3 serotypes as well bind with Laminin receptor.
DEN 2–4 serotypes also bind with an unknown protein having the property of serotype specific binding. After initial attachment of the virus with particular receptors on th

10. The viral particle is fused into acidic lysosomes through receptor-mediated endocytosis.
After that, viral particle is uncoated and the RNA is released in host cell
RNA is released in host cell where it directs the synthesis of viral proteins.
Once all the essential proteins are synthesized, viral RNA starts copying to generate a minus strand, which is then transcribed to new plus stranded molecules.
In only few hours after infection, tens of thousands copies of viral molecules are produced from a single viral molecule leading to cell damage and in severe cases to death. 

12. Viral Pathogenesis

13. Viral Pathogenesis

14. Viral Pathogenesis

15. Role of ADE in Dengue

16. Immune response to DENV

18. Bunyaviridae infections
Arthropod vector associated viral infections;
Exception – Hantaviruses
RVF – Aedes mosquito
CCHF – Ixodid tick
Hantavirus – Rodents
Less common
Aerosol
Exposure to infected animal tissue

19. Bunyaviridae in Animals
RVF
Abortion – 100%
Mortality rate
>90% in young
•5-60% in older animals
• CCHF
• Unapparent infection in livestock
• Hantaviruses
• Unapparent infection in rodents

20. Transmission of CCHF Disease
CCHFV usually circulates between asymptomatic animals and ticks in an enzootic cycle.
This virus has been found in at least 31 species of ticks, including seven genera of the family Ixodidae (hard ticks).
Members of the genus Hyalomma seem to be the principal vectors. Transovarial, transstadial and venereal transmission occur in this genus.
Hyalomma marginatum is particularly important as a vector in Europe, but CCHFV is also found in Hyalomma anatolicum anatolicum andother Hyalomma spp.

21. Bunyaviridae
CCHFV is a member of the Nairovirus genus of the family Bunyaviridae. Other genera within the family include Orthobunyavirus, Hantavirus, Phlebovirus, and Tospovirus.
there are seven recognized species in the genus Nairovirus containing 34 viral strains. The most important Serogroups are the CCHF group, which includes CCHFV, and Hazara virus, which has not been demonstrated to be pathogenic to human
hemagglutination inhibition (HI), complement fixation (CF) and agar gel diffusion and precipitation (AGDP) tests have shown the virus to be antigenically related to no other viruses except: to Hazara with which it constitutes the CCHF group

22. CCHF is a Tick borne Disease
CCHF spreads to humans either by tick-bites, or through contact with viraemic animal tissues during and immediately post-slaughter.
CCHF outbreaks constitute a threat to public health services because of its epidemic potential, its high case fatality ratio (10-40%), its potential for nosocomial outbreaks and the difficulties in treatment and prevention
Clinical disease is rare in infected mammals, but commonly severe in infected humans, with a 30% mortality rate.
Outbreaks of illness are usually attributable to handling infected animals or people.

24. CCHFV

25. CCHFV
Crimean–Congo hemorrhagic fever virus has a tripartite genome consisting of large (L), medium (M), and small (S) negative-sense RNA segments that encode the viral RNA polymerase, the glycoproteins, and the nucleocapsid protein (NP), respectively (Clerx et al., 1981).
Viral infection is detected by host cells via Toll-like receptor (TLR)-dependent and -independent mechanisms which results in ubiquitination and ISGylation of target proteins to induce the innate immune system.
Ubiquitination involves a regulatory protein called Ubiquitin (Ub) which conjugates with target (viral) proteins and forms an Ub-tag which in turn, recognized by proteosome for destruction

26. Crimean–Congo hemorrhagic fever virus infection cycle
Crimean–Congo hemorrhagic fever virus infection cycle. L, M, and S, viral genomic segments; GPC, glycoprotein complex; TLR, Toll-like receptor; RdRp, RNA-dependent RNA polymerase; OTU domain, ovarian tumor domain; IFN, interferon; Ub, ubiquitin; ISG15, IFN simulated gene product 15.

27. Virus life cycle

28. Symptoms Fever, fatigue, dizziness, myalgia's, and prostration
Signs of bleeding range from only conjunctival hemorrhage, mild hypotension, flushing, and petechiae to shock and generalized mucous membrane hemorrhage and evidence of pulmonary, hematopoietic, and neurologic dysfunction
Renal insufficiency is proportional to cardiovascular compromise except in Hemorrhagic Fever and Renal Syndrome in which it is an integral part of the disease
severe bruising, severe nosebleeds, and uncontrolled bleeding at injection sites can b seen, beginning on about the fourth day of illness and lasting for about two weeks

30. Symptoms and Diagnostics
The liver becomes swollen and painful.
Disseminated intravascular coagulation may occur as well as acute kidney failure and shock, and sometimes acute respiratory distress syndrome
Laboratory diagnosis of CCHF can be made by finding a positive serological test result,
evidence of viral antigen in tissue
by immunohistochemical staining and microscopic examination,
identification of viral RNA
sequence in blood or tissue, in a patient with a clinical history compatible with CCH
At autopsy, the virus is most likely to be found in the lung, liver, spleen, bone marrow, kidney and brain.

31. serology.
Crimean-Congo hemorrhagic fever can be diagnosed by serology.
Tests detect CCHFV-specific IgM, or a rise in IgG titers in paired acute and convalescent sera.
IgG and IgM can usually be found with mindirect immunofluorescence or ELISA after 7-9 days ofillness