Lecture 12

Flaviviruses

Overview of Flavivirus Family

• The family of Flaviviruses includes several genera:

  • Flaviviruses (e.g., Yellow Fever, West Nile, Dengue)

  • Pestiviruses (e.g., Bovine Viral Diarrhea Virus (BVDV), Classical Swine Fever Virus (CSFV))

  • Hepacivirus (e.g., Hepatitis C Virus (HCV))

• Key characteristics:

  • Positive single-stranded RNA genome approximately 11 kb in length.

  • The RNA genome is capped and NOT polyadenylated.

  • Naked RNA is infectious, meaning the RNA can initiate infection directly.

  • The genome is translated into a single polyprotein, which is processed into ten individual proteins by cellular and viral proteases.

Structural Characteristics of the Flavivirus Genome

• Structural proteins include:

  • C: Capsid protein

  • prM/M: Pre-membrane protein/Membrane protein

  • E: Envelope protein

• Nonstructural proteins include:

  • NS1: Secreted and involved in immune evasion.

  • NS2A: Localized to the ER membrane and involved in persistence.

  • NS2B: ER membrane; cofactor for NS3 protease.

  • NS3: ER membrane; functions as a protease and helicase.

  • NS4A: ER membrane; involved in replication and pathology.

  • NS4B: ER membrane; has roles in interferon inhibition.

  • NS5: ER membrane protein; serves as the RNA-dependent RNA polymerase (RdRP).

Lifecycle of Flavivirus

• The lifecycle can be described in several steps:

  1. Binding to the host cell receptors.

  2. Receptor-mediated endocytosis occurs, leading to internalization of the virus.

  3. Low pH-dependent membrane fusion occurs to release the viral genome.

  4. Uncoating of the RNA genome.

  5. Translation & polyprotein processing takes place in the host cell's cytoplasm.

  6. Membrane-associated RNA replication occurs.

  7. Virion morphogenesis happens in intracellular vesicles.

  8. Virion transport to the plasma membrane.

  9. Vesicle fusion at the plasma membrane, resulting in virion release.

Flavivirus Diseases

• Flaviviruses are responsible for various diseases, including:

  • Fever syndromes

  • Encephalitis conditions

  • Hemorrhagic fevers

• Major global concerns include:

  • Dengue Virus (DENV)

  • Yellow Fever Virus (YFV)

  • Japanese Encephalitis Virus (JEV)

  • Recently, the Zika virus has garnered attention.

• Regional or endemic concerns: St. Louis Encephalitis, West Nile virus, Tick-borne encephalitis virus.

• Some flaviviruses like Hepatitis C are non-vector borne.

• Available vaccines include those for YFV, JEV, TBEV, and pestiviruses (CSFV and BVDV).

Transmission and Infection Dynamics of Arthropods

• Arthropod Infection Mechanism:

  • Infection cycle includes:

  1. Ingestion of infectious blood meal by mosquito.

  2. Virus infects mesenteron epithelial cells and multiplies.

  3. Virus is released from mesenteronal epithelial cells into the mosquito hemocoel.

  4. Virus can either:

     • a. Infect salivary glands after secondary amplification.

     • b. Infect salivary glands directly without prior amplification.

  5. Virus is transmitted to the host from salivary gland epithelial cells during feeding.

• Key barriers include:

  • Midgut infection barrier

  • Midgut escape barrier

  • Salivary gland infection barrier

Experimental Procedures on Mosquito Infection

• Oral Infection Studies:

  • Mosquitoes were fed an artificial blood meal with $3 imes 10^8$ infectious units/ml of WNV single-cycle infectious particles.

  • Midguts analyzed for WNV antigen expression on days 3-8 post-infection.

• Secondary Organ Infection Assessment:

  • Goal: Determine the tropism of WNV in secondary organs and susceptibility to infection with viral-like particles (VLPs).

  • Method: Intrathoracic inoculation performed to bypass barriers.

  • Analysis: Harvest fat body, midgut, and salivary glands post-infection; detect WNV antigen using immunofluorescence assay and confocal microscopy.

Observations from Intrathoracic Inoculations

• Samples reveal:

• Opportunistic infection of organs like fat body, tracheal cells, and the midgut muscle, with notable susceptibility feedback hierarchy:

  • Fat Body > Tracheal Cells > Midgut Muscle > Salivary Glands.

• Direct infection of secondary organs can occur if hemocoel titers are sufficiently high.

Flavivirus Pathogenesis Observations

• Flaviviruses may replicate in or around the inoculation site but often infect Langerhans dendritic cells (DCs) in the skin.

• These DCs move to the draining lymph nodes where:

  • DCs present antigens to other immune cells and secrete proinflammatory cytokines to limit or clear the infection.

  • Virus replication in infected DCs aids its spread through the lymphatic system.

• Other organ infections are less characterized and vary by virus.

Dengue Fever Overview

• Dengue virus comprises four serotypes (DENV 1-4).

• Annual incidence: 100 million cases worldwide, with 2.5 billion people at risk.

• Transmission occurs predominantly via Aedes aegypti mosquitoes in urban areas.

• Historical control methods included the Pan American Health Organization's successful eradication in the 1950s-60s.

• Dengue reinfestation occurred in the 70s, with cases re-emerging in the Caribbean and Pacific in the last two decades.

Global Distribution Changes in Dengue Virus Serotypes (1970 - 2004)

• 1970 and 2004 distributions depicted changes in global serotype prevalence, highlighting shifts in viral strains over the years in response to human-mosquito dynamics.

Dengue Pathogenesis

• Disease outcomes vary:

  • Classic dengue presents as self-limited infection.

  • Severe dengue presentations include increased vascular fragility, coagulopathy, and potential for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS).

  • Initial symptoms include a sudden onset of fever, severe headache, retro-orbital pain, myalgia, and rash.

• Antibody-dependent enhancement (ADE) observed in secondary infections can exacerbate disease severity.

• Subsequent infections with heterologous serotypes can lead to increased viral loads and severe manifestations due to antibody-mediated uptake by mononuclear cells.

Yellow Fever Overview

• Epidemiology similar to dengue, transmitted in urban environments by Aedes aegypti mosquitoes.

• The successful mosquito eradication program drastically reduced cases to 150 in the 60s, but resurgence occurred in the 90s with thousands of annual cases.

• Pathological effects are primarily seen in the liver, leading to hepatocyte death and mild inflammation, with resultant hemorrhagic fever.

• Clinical symptoms can mirror dengue fever, including jaundice and bleeding manifestations.

• Mortality in severe cases can reach 20-50%, typically occurring 7-10 days post-illness onset.

Yellow Fever Vaccine

• The YFV 17D vaccine is derived from a live attenuated virus.

  • Developed through 176 passages of the wild-type strain Asibi in mouse and chicken tissue.

  • Over 500 million doses administered since its inception in 1937.

West Nile Virus (WNV) Overview

• Initially isolated in Africa, WNV became a significant public health issue following its emergence in the US in 1999.

• Associated with severe cases of encephalitis and fatalities, particularly in the elderly.

• Transmission involves a cycle with specific Culex mosquitoes; humans do not serve as maintenance hosts.

WNV Clinical Manifestations

• Majority of cases are asymptomatic, but symptomatic cases may result in West Nile fever, presenting with:

  • Sudden fever onset

  • Malaise, nausea, anorexia, headache, and rash.

• Neurological complications can develop in elderly patients, leading to conditions like West Nile encephalitis or acute flaccid paralysis.

• Reference: Petersen, L, et al. JAMA, 2003; symptomatic cases are roughly ~20% of those infected.

Hepatitis C Virus (HCV) Overview

• HCV was identified as the non-A, non-B hepatitis pathogen (NANBH).

• Blood-borne transmission with capability for chimpanzee infection.

• HCV belongs to the family Flaviviridae, specifically the genus Hepacivirus, which is the only member of its genus.

HCV Genome Structure

• Approximately 9,600 bases of positive-stranded RNA with various functional gene regions.

• The genome includes unique elements such as an IRES at the 5' end.

Pathogenesis of HCV Infection

• Primary replication occurs in the liver, leading to conditions such as steatosis (fatty degeneration of hepatocytes), fibrotic changes, cirrhosis, and potentially hepatocellular carcinoma (HCC).

• Immune-mediated injuries result from chronic inflammation initiated by HCV-specific T cells, contributing to liver pathology.

• Notably, about 85% of infected individuals experience chronic infection lasting beyond six months.

Histopathological Changes in HCV-Related Disease

• Key pathological features include:

  • Lymphoid aggregates

  • Steatosis (fat accumulation)

  • Fibrosis marked by collagen deposition.

  • Advanced fibrosis indicating extensive scarring in liver tissue.