• 45ml of viral diluent
• 1 vial of 0.5ml kunjin virus suspension
• Sterile test tubes
• Sterile pipette
• 2 6-well trays of confluent BHK-21 cell
• 96ml of viral diluent
• 60ml of 2times concentration maintaince medium
• 60ml of 2% CMC aquacide II Solution in ultrapure water
• Beaker for discard
  

• Laboratory animals-Apes and Monkeys, Mouse
• Chick Embryo
• Tissue Cell Culture
  1. Primary cell cultures
  – Heterogeneous – many cell types
  – Closest to animal
  – Technical hassle
  
  2. Diploid cell strains
  –Relatively homogeneous – fewer cell types
  – Further from animal
  – Technically less hassle
  
  3. Continuous cell lines
  – Immortal
  – Most homogeneous
  – Genetically weird – furthest from animal
  – Hassle free
  – Suspension or monolayer
  l Tissue Culture Methods for Detection
  1. Cytopathic effect (CPE)
  2. Plaque Assay

Physical Methods

• Hemagglutination
• Immunological tests for proteins
• Assay for nucleic acid (Southern, PCR)
• Enzymatic (reverse transcriptase for retroviruses)
• Comparison of quantitative methods
• Serological/ Immunological Methods
  - Hemagglutination (HA)
  - Hemagglutination Inhibition (HI)
  - Virus Neutralization,
  - Complement Fixation
  - ELISA, RIA, LA, western blot
  
  Specimens for viral diagnosis

The Small Pox Virus

Morphology

• Ovoid and brick-shaped
  
• 160-190nm in diameter, 220-450nm in length
  
• Virions - envelope, surface membrane, a core and lateral bodies
  
• Virus capsid is enveloped
• External Coat - lipid and tubular or globular protein structures enclosing lateral bodies

Genome

• Linear double-stranded DNA
  
• 130000-375000 nucleotides long
• Sequences repeated at both ends
• Double-stranded DNA - Cross-linked at both ends

Diseases

• Smallpox
• Variola
• Vaccinia
  
• Cowpox
  
• Monkeypox

Smallpox

• Transmit from human to human, no animal reservoir or insect that play a part in transmitting.
  
• Virus transmit through infected aerosols and water droplets in face to face contact.
  
• Can also be transmitted through the clothes, belonging etc of infected person. However chances are lower.
• have an incubation period of 7-17days
  
• initial sign and symptoms are quite similar to influenza
• Variola major - 25-30% fatalities. Do not spread that widely because patient is unable to leave bed during the early phase and stay in bed through out the illness.
  
• Variola minor - less than 1% death. Spread widely and more easily because infected person is still able to continue with daily actives in early phase.
  
• Before vaccine was developed, infected people either die or survive but heavily scarred, became blind and suffer neurological damages.
  
• Vaccination was then developed by Edward Jenner (the video below tells you more)

• Although Vaccine was developed in the 1800s but eradication was only declared in 1980,
• In the early face of vaccination, the death rate of small pox did not decreas but increased.
  
• Many that were vaccinated made themselves in a higher risk of being infected by smallpox and other diseases because of many factors
• the vaccine was then further improved and then lead to the eradication

• Eradication was possible because the virus can only infect human but no reservoir was there
• Most at that time were protected by the vaccine or was infected and have a life long immunity to smallpox.
  

More than 100 different members of the Flaviviridae are known, and are subdivided into 3 genera:
I. Flavivirus (including dengue, yellow fever, and West Nile viruses)
II. Pestivirus
III. Hepacivirus (hepatitis C viruses)

These genera have diverse biological properties and cause distinct diseases. However, they have similar genome organization and replication mechanisms.

Virion Properties

Morphology

• Virions consist of an envelope and a nucleocapsid
• Virus capsid is enveloped
• Virions are spherical to pleomorphic measuring 40-60 nm in diameter
• Surface projections are small spikes surrounded by a prominent fringe
• Capsid/nucleocapsid is round and exhibits polyhedral symmetry. The core is isometric and has a diameter of 25-30 nm

Genome

• Monopartite, linear, ssRNA(+) genome of about 10-12 kb
• Virion RNA is infectious, serves as both the genome and the viral messenger RNA
• Has 5' end encodes structural proteins which has a stem and loop structure
• Non-structural proteins (protease, helicase, polymerase) encoded at the 3' end

Examples of Flavivirus

Dengue Virus
Dengue virus causes dengue and dengue hemorrhagic fever. It is an arbovirus. It has four serotypes, known as DEN-1, 2, 3, and 4. Most common caused by Aedes aegypti Mosquito.

Aedes aegypti can be identified by the white bands or scale patterns on its legs and thorax.
Two electron images of mature Dengue-2 virus particles replicating in five-day-old tissue culture cells:


Dengue Fever (Primary Infection)

Dengue fever is an acute viral illness characterized by:

• Fever
• Headache
• Muscle and joint pain
• Nausea/vomiting
• Rash
• 
• Hemorrhagic manifestations

Some cases may present with or develop encephalitic signs and symptoms, such as:

• Decreased level of consciousness—including lethargy, confusion, and coma
• Seizures
• Nuchal rigidity
• Paresis
• Dengue Haemorrahgic Fever / Dengue Shock Syndrome (Secondary Infection)

Four Grades of DHF

1. Grade 1
   Fever and nonspecific constitutional symptoms
   Positive tourniquet test is only hemorrhagic manifestation
2. Grade 2
   Grade 1 manifestations + spontaneous bleeding
3. Grade 3
   Signs of circulatory failure (rapid/weak pulse, narrow pulse pressure, hypotension, cold/clammy skin)
4. Grade 4
   Profound shock (undetectable pulse and BP)
   Symptoms

Antibody Response

Infection will result in lifelong immunity to that serotype, but only temporary immunity to other serotypes.

Primary Infection

• IgM antibodies appear approximately 5 days after onset of symptoms and rise for the next 1-3 weeks
• IgM antibodies detectable for up to 6 months
• IgG are detectable at approximately 14 days after onset of symptoms and are maintained for life

Secondary Infection

• Approximately 5% patients do not produce detectable levels of specific IgM
• IgM titre can be slower to rise in secondary infection
• IgG appears approximately 2 days after symptoms appear
• IgG titre significantly higher in secondary infection

Treatment

• No specific treatment for dengue fever.
• Intravenous fluid replacement and use of plasma expanders oxygen therapy blood transfusions in cases of severe bleeding heparin for severe haemorrhage.

Prevention

Vaccination???
Presently no vaccine for prevention.
Vaccine development for dengue and DHF is difficult because any of four different viruses may cause disease, and because protection against only one or two dengue viruses could actually increase the risk of more serious disease.

Vector Control:
Chemical Control

• Insecticide
• Mosquitoes screen

Biological and Environmental Control

• Place fish in containers to eat larvae
• Elimination of larval habitats

Yellow Fever Virus

Yellow fever is transmitted by the bite of an infected mosquito. Infants and children are at higher risk. There are two cycles of infection: one carried by monkeys and one by humans.

Jungle yellow fever

• Mainly a disease of monkeys in the tropical rain forest
• People get it when they are bitten by mosquitoes that have been infected by monkeys
• Is rare and occurs mainly in persons who work in tropical rain forests

Urban yellow fever

• A disease of humans
• Spread by mosquitoes that have been infected by other people
• Cause of most yellow fever outbreaks and epidemics

Symptoms

Yellow fever's incubation period (the amount of time between the introduction of the virus into the host and the development of symptoms) is three to six days. During this time, there are generally no symptoms identifiable to the host.

Period of invasion
-lasts two to five days
-begins with an abrupt onset of symptoms

• Fever and chills
• Intense headache
• Muscle aches
• Nausea
• Strawberry tongue

Period of intoxication
-last 3 to 9 days
-represents the most severe and potentially fatal phase of the illness)

• Jaundice
• Black vomit
• Hypotension
• Shock
• Cardiac arrhythmia
• Confusion

Treatment

No specific treatment for yellow fever.
Dehydration and fever can be corrected with oral rehydration salts and paracetamol. Any superimposed bacterial infection should be treated with an appropriate antibiotic.

Prevention

Vaccination???
A very safe, very effective yellow fever vaccine exists. About 95% of vaccine recipients acquire long-term immunity to the yellow fever virus.
Vector Control:
· Insecticide

West Nile Virus


West Nile fever is a disease caused by West Nile Virus. West Nile Virus has an extremely broad host range. It replicates in humans, horses, and several species of birds. Most infected individuals show few signs of illness, but some develop severe neurological illness which can be fatal.

Symptoms
Most cases are mild and flu-like symptoms:

• Fever
• Muscular aches and pains
• Rash
• Swollen lymph glands

In more severe cases:

• Encephalitis
• Meningitis
• Confusion
• Severe muscle weakness

Typically those patients who develop a more severe form of the disease are likely to be people with a weakened immune system which includes the older person or those with certain chronic illnesses.

Treatment

No specific treatment, other than supportive care, is available.

Prevention

Vaccination???
No vaccine is available.

Vector Control:
Insect repellents

• Alpharetrovirus
• Betaretrovirus
• Deltaretrovirus
• Epsilonretrovirus
• Gammaretrovirus
• Lentivirus

Spumaretrovirinae (subfamily)

• Spumaretrovirus

Virion Properties
Morphology

• Consist of an envelope, a nucleocapsid, and a nucleoid
• Virus capsid is enveloped
• Virions are spherical to pleomorphic
• Virions measure 80-100 nm in diameter
• Surface projections are densely dispersed, small or distinctive glycoprotein spikes that cover evenly the surface
• The nucleoid is concentric, or eccentric

• Dimeric (monomers held together by hydrogen bonds)
• Unsegmented and contains a single molecule of linear
• -RT and a positive-sense, single-stranded RNA
• One monomer is 7000-11000 nucleotides long
• The encapsidated nucleic acid is mainly of genomic origin, but virions may also contain nucleic acid of host origin
  - including host RNA and fragments of host DNA believed to be incidental inclusions

All retroviruses contain three large genes which are, reading from 5’ to 3’:

i. gag - contains information to direct the synthesis of internal virion proteins that form the matrix, the capsid and the nucleoprotein structures

ii. pol - contains information for the reverse transcriptase and integrase enzymes

iii.env - contains the information for building the surface and transmembrane components of the viral envelope protein.

Examples of Retrovirus

Human T-cell lymphotropic virus (HTLV)
There are four different types of human T-cell lymphotropic viruses:
HTLV-1 : HTLV-1 has an affinity for CD4+ T-cells and is associated with several types of disease states: Adult t-cell leukemia, tropical spastic paraparesis, uveitis, and dermatitis.

HTLV-2: HTLV-2 mainly infects CD8+ T-cells, but does not have any proven causative role in human lymphoproliferative diseases.

HTLV-3: HTLV-3 was originally isolated in association with AIDS. With further research, however, it was discovered that its pathogenic and genetic characteristics differed from those of HTLV-1 and HTLV-2.

HTLV-4: HTLV-4 has only been described in cases involving bushmeat hunters in Africa.

An infected T-Cell with HTLV-1(green)

• sexual activity
• sharing used needles or syringes
• mother-to-child transmission through childbirth or breast feeding
• receiving transfusions of infected blood, transplanted organs or donated sperm
  

Symptoms
Stages of HIV infection has been broken down into four steps:

A. Primary Infection
During stage 1, there is a short flu-like illness that is often mistaken for being nothing serious, thus diagnosis is frequently missed. This stage generally lasts for a few weeks, during which the immune system attempts to fight back by producing HIV antibodies and cytotoxic lymphocytes.

• swelling of the lymph nodes
• headache
• fever
• loss of appetite
• sweating
• sore throat

B. Clinically Asymptomatic Stage
Even though no symptoms are present, the virus is multiplying (or making copies of itself) in the body during this time. HIV multiplies so quickly that the immune system cannot destroy the virus. After years of fighting HIV, the immune system starts to weaken.

• Weight loss
• Fungal Nail Infections
• Repeated outbreaks of herpes simplex
• Personality changes
  

C. Symptomatic HIV Infection
It occurs when the immune becomes severely damaged by HIV. The body has been fighting back for years, the virus could mutate and become stronger and the T helper cells are dying off, with turnover rates failing to keep up.

• Severe weight loss
• Severe bacterial infections
  

D. Progression from HIV to AIDS
As the immune system becomes more and more damaged the illnesses that occur become more and more severe leading eventually to an AIDS diagnosis.
Kaposi's sarcoma (a skin tumor that looks like dark purple blotches)

• Shortness of breath and difficulty breathing due to infections of the lungs
• Dementia
• Severe malnutrition
• Chronic diarrhea
  

Treatment

Antiretroviral Drugs
Block the activity of one of the enzymes HIV needs to replicate inside human cells.

These drugs are:

• nucleoside reverse transcriptase inhibitors (NRTIs) / non-nucleoside reverse transcriptase inhibitors (NNRTIs)
  -interfering with the action of reverse transcriptase.
• protease inhibitors
  - inhibit protease which cleaves the polyprotein to yield active virions
• fusion or entry inhibitors
  - prevent HIV from entering human cells

Treatment is most effective when at least two or three drugs are given in combination, referred to as highly active antiretroviral therapy (HAART). It can delay or prevent AIDS in HIV-infected people, thus extending their life.

Diagnosis
Specific tests include the following:

• Enzyme-linked immunosorbent assay (ELISA)
• Newer rapid screening tests
• Western blot