MIMM 465 Lecture Notes - Lecture 4: Multiplex Polymerase Chain Reaction, Glycoprotein, Differential Centrifugation

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Published on 16 Apr 2013
School
McGill University
Department
Microbiology and Immun (Sci)
Course
MIMM 465
Lecture 4
<Henipavirus Dr. Briedis>
Henipavirus
- A genus of the family Paramyxoviridae, order Mononegavirales
- Contains 3 established species:
1. Hendra virus
2. Nipah virus
3. Cedar virus
- Example of emergent virus diseases
HIV and SARS was also an emergent virus disease
Cause serious disease and not have been known in people
In Brisbane, Australia…
- The center of Australian horse racing
- There are many horse farms
- In August 1994, Mackay, Australia (1,000km north of Brisbane)
Two horses developed frothy nasal discharge and subsequently DIED
Their owner, Mark Preston, assisted in necropsies (autopsies) of the horses
Within 3 weeks the owner was admitted with meningitis initially recovered 14 months lather developed
neurologic signs and died
This outbreak wasn‟t initially diagnosed as being caused by any specific virus
- In September 1994 Hendra, a suburb of Brisbane in Queensland, Australia
The index case, a mare was housed with 19 other horses after falling ill, and it died 2 days later
Subsequently, all of the horses became ill, with 13 dying
The head trainer, Vic Rail, and a stable hand were involved in nursing the index case
BOTH fell ill with influenza-like illness w/in 1 week of the 1st horse‟s death
The stable hand recovered while Mr. Rail died of respiratory and renal failure
The source of the virus was most likely frothy nasal discharge from the nose of the index case
- The 6 surviving horses were quarantined
Body fluid samples taken
Then the surviving horses were euthanized as a way of preventing relapsing infection and possible further
transmission
Samples were transferred to the center for disease control and prevention (CDC)
- Level 4 laboratory
- They 1st tried to identify the virus by multiplex PCR
Multiplex PCR
- Four or more different are ran in a single reaction, each of them giving you a specific band
- You are doing a PCR for a likely virus candidate, such as coronavirus, influenza, etc in a rapid fashion
- They did this for ~38 viruses and came out COMPLETELY negative
Rapid ID of emerging infectious agents using PCR and electrospray ionization mass spectrometry
- To identify product bands
- Automated PCR/ESI-MS biosensor
- Key modules include
Amplicon purification (desalting)
Plate stackers so you could run different plates (each plate has a well that you could run a particular PCR
rxn) can run 64 wells in each of the plate
Automated sample injection and mass spectrometry analysis
- Precise molecular weight determinations of amplicons yield unambiguous base compositions that are used to
uniquely “fingerprint” each pathogen
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- They take primers
that are homologous
to the most conserved
regions of every
known virus group &
multiple conserved
regions To identify
new viruses
- The automated
system is capable of
analyzing more than
1,500 PCR rxns in
24h
- They had to run
12,000 PCR rxns
before nailing down
the new virus
- W/in 6 days the
CDC IDed the
causative agent in Hendra, Australia as a hitherto unknown Paramyxovirus
This was named Hendra virus
Like a typical Paramyxovirus, it has a plasma membrane with surface glycoprotein spikes in it, has
nucleocapsid, and has long single stranded (15,000 nts) RNA that is encapsidated in a (-) sense
The virus has 2 surface proteins, F (fusion) protein and G (attachment) protein
- G protein binds to host cell receptor
Matrix (M) protein underlies the plasma membrane, associated with genome ssRNA
Nucleocapsid transcriptase complex that consists of L (large polymerase) protein, N (nucleocapsid) protein and
P (phospho) protein
Gene map of Paramyxovirus
- Starting from the 3‟ end of the (-) strand of genome RNA, there are 6 cistrons, encoding the 6 known proteins
that are encoded by the virus
The Hendra virus G protein was expressed in a yeast vector
- They removed the carboxy-terminal anchor region and the yeast secreted soluble G protein (which was glycosylated
by the yeast) into the tissue culture medium that the yeast was grown in
- The resulting large amount of secreted G protein were purified by chromatography were used as antigen to set up
an ELISA assay for specific serum antibody against Hendra virus
Started to look for WHERE the Hendra virus came from
- Emerging virus come from either: isolate human populations that suddenly came into contact with rest of
humanity (not common) OR from animal reservoir that suddenly came into contact with human
- The ELISA was used to screen 46 species of wildlife in the vicinity of Hendra, Australia
The only seropositive culprit came out to be a fox-headed fruit bat!
Fox-headed fruit bat
- BIG wings spread about 3feet
- Have faces that look like foxes and body size is close to foxes
- Fly BOTH at day and night
- Hang from trees
- QUITE a lot of them in North-eastern Australia (near Hendra)
- Eat fruits and more commonly drink flower nectar like insects
Originally called vampire bats, b/c during the night time, you could hear the sucking noises of the plant nectars
- BAT saliva carries viruses
As of December 2012, 39 outbreaks of Hendra virus have occurred in Australia
- ALL in North-eastern Australia
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Document Summary

A genus of the family paramyxoviridae, order mononegavirales. Contains 3 established species: hendra virus, nipah virus, cedar virus. Hiv and sars was also an emergent virus disease. Cause serious disease and not have been known in people. In august 1994, mackay, australia (1,000km north of brisbane) Two horses developed frothy nasal discharge and subsequently died. Their owner, mark preston, assisted in necropsies (autopsies) of the horses. Within 3 weeks the owner was admitted with meningitis initially recovered 14 months lather developed neurologic signs and died. This outbreak wasn t initially diagnosed as being caused by any specific virus. In september 1994 hendra, a suburb of brisbane in queensland, australia. The index case, a mare was housed with 19 other horses after falling ill, and it died 2 days later. Subsequently, all of the horses became ill, with 13 dying. The head trainer, vic rail, and a stable hand were involved in nursing the index case.

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