Newcastle Disease Flashcards
What are the basic features of Newcastle disease?
Enveloped, pleomorphic, up to 500 nm Single-stranded, negative sense RNA genome Genome is 15 kb Replicates in the cytoplasm Causes lytic infection in cells
What is the taxonomy of Newcastle disease?
Order: Mononegavirales
Family: Paramyxoviridae
Genus: Avian orthoavulavirus- 1
What is the serotype of Newcastle disease?
Type species of avian orthoavulavirus serotype-1
What do all AOaV-1 strains belong to?
They all belong to a single serotype, but there are over 18 genotypes
What are the six structural proteins in the NVD?
1) Nucleoprotein (NP)
2) Phosphoprotein (P)
3) RNA dependent RNA polymerase (L)
4) Matrix (M)
5) Hemagglutinin-neuraminidase (HN)
6) Fusion (F)
What is the non-structural protein in the NVD?
1) V
Inhibits interferon
Not packaged within virion
There is also the RNA genome
What is the Ribonucleoprotein?
RNP: replicative unit of the virus
How does the RdRp act within the NDV?
RNA-dependent RNA polymerase engages NP- encapsidated RNA
What is the phosphoprotein?
The phosphoprotein is a cofactor of the polymerase
Explain the polymerase in NDV?
The polymerase can act as the replicase, which produces copies of the viral gene
The polymerase can act as a transcriptase, which makes mRNA for viral proteins
How does transcription of NDV work?
1) The polymerase binds the genome at the leader sequence
2) Transcription is sequential, which means it starts from the 3’ end and produces separate mRNAs as it progresses downstream. Genes are transcribed by recognizing start and stop signals flanking viral genes
3) mRNAs are capped and polyadenylated
4) The V protein is produced by post transcriptional insertional frameshift in the transcript of the P gene
What is transcription in NDV?
It is polar
Transcriptional units at the 3’ end are more transcribed than those at the 5’
What are the transcriptional units in NDV?
Transcripts 1, and 3-6 are monocistronic, the remaining are bicistronic
There is a start, regulatory sequences (UTRs), Coding region, UTR, and an end
What is the genome of NDV?
3’- Leader - NP - P-V- M - F -HN - L
What is the infectious cycle of NDV?
1) NDV attaches to the N-glycosilated receptors on the plasma membrane through the HN on the NDV
2) Fusion of the NDV is mediated by the F protein
3) Release of RNP into the cytosol
4) Genome replication mediated by L protein, transcription mediated by L protein
5) Translation
6) Virus assembly
7) Release mediated by HN
How does the release mediated by HN occur?
It occurs at lipid rafts, which is where the virus will be released from the cytoplasm
What are the lipid rafts?
The lipid rafts are cholesterol rich areas of the plasma membrane
Cytolytic cycle
The entire infectious cycle occurs in the cytoplasm
What is the HN protein?
Tetramer on the surface of the virus
Allows attachment of virion to N-glycosylated protein on the plasma membrane (hemagglutinin activity)
Allows release of mature virions (neuraminidase activity )
Hemagglutination activity used for titration
What is the F protein?
Trimer on the surface of the virus
Produced as F0, so needs to be cleaned to be activated F1 + F2
Allows fusion of the virus envelope to the plasma membrane to release the RNP, which enables infection
How does the F protein contribute to virulence?
If the F protein has a poly basic configuration of the cleavage site, F0 can be cleaved in every tissue, which leads to systemic spread and severe disease, hence virulent strains
If the F protein has a non poly basic configuration of the cleavage site, F0 can be cleaved only in the intestine and respiratory tract, localized infection and non-virulent strain
Explain how the poly basic fusion cleavage site affects the bird?
Systemic virus replication in multiple tissues (virulent NDV) bird will die
Explain how the mono basic fusion cleavage site affects the bird?
Local virus replication in respiratory tract and intestine (non virulent), this is self-limiting so the bird will not die
What is most susceptible to NDV?
Poultry
What are the four patho types of NDV?
Velogenic, mesogenic, lentogenic, asymptomic enteric
Explain the velogenic strain of NDV
Has a poly basic cleavage site, up to 100% morbidity and mortality, and is reportable in Canada
Explain the mesogenic strain of NDV
Has a poly basic cleavage site, moderate morbidity and mortality, respiratory lesions usually complicated by secondary infection, not reportable in Canada
Explain the lentogenic strain of NDV
Does not have a poly basic cleavage site, minimal respiratory disease, usually complicated by bacterial pathogens, not reportable in Canada
Explain the asymptomatic enteric strain of NDV
Does not have a poly basic cleavage site, there are no clinical signs or lesions, and not reportable in Canada
What is special about the lentogenic strain of NDV?
Used as a vaccine for velogenic and mesogenic because all strains do have the same serotype
This strain is also used in the laboratory
How is virulence differentiated in chicks?
Let’s say we have a group of 10 chicks that are less than 48 hours old –> Need to be less than 48 hours old
These chicks are inoculated intracranially with 50 uL of virus inoculum
Scored for 8 days: 2 died, 1 is sick, and 0 normal, so
the final score becomes 0(mild) - 2 (severe)
What does the NDV velogenic strain cause?
Necrosis, hemorrhagic, death, neurological signs (including paralysis and tremors)
How is algorithm tested for NDV?
1) Suspect or presumptive outbreak (veterinarian observes high mortality, positive molecular test in regional laboratory)
2) Confirmation by CFIA (PCR)
3) Outbreak is declared
What happens when an NDV outbreak is declared?
1) Depopulation of the area
2) Primary control zone:
Infected zone: 3 km within outbreak, quarantine, quarantine can be lifted after 21 days of negative tests
Restricted zone: 3-10 km, constant screening
Security zone: >10 km
3) Trade restrictions
How are eggs used to propagate NDV?
Embryonic chicken egg that is 9-10 days old.
Chorioallanotic cavity
Eggs are kept for 5 days, allantoic fluid collected for additional tests
Can get up to 10^7 infectious viral units/ml
How are cells used to propagate NDV?
NDV can be isolated and propagated in several cell lines (DF-1, CEF, VERO, Hep-2)
Lentogenic strains require trypsin supplementation to cleave the F protein and allow infection
Virulent strains do not require trypsin
Development of cytopathic effect
How is titration used in NDV?
Hemagglutination assay
Plaque formation
Tissue culture or embryo infectious dose 50%
What is NDV reverse genetics?
Creation a virus from a full length cDNA copy of the viral genome
Very powerful technique
Allows to introduce foreign genes
Allows gain or loss of function experiments to understand the molecular determinants of certain phenotypes (virulence)
What is virus rescue?
The full cDNA clone (infectious) is cloned into an expression plasmid under the T7 RNA polymerase promoter
FLC which is the cDNA clone is co-transfected in the cells with the plasmids expressing the coding sequences under T7 promoter
The NP, P, L –> are part of the helper plasmid under T7 promoter
Why is NDV an excellent vaccine vector?
The NDV genome can be easily manipulated
First recombinant NDV expressing a foreign gene was reported in 2000
NDV can be propagated easily in cells and embryonated chicken eggs
NDV can accommodate and express a foreign gene in a stable manner
Low risk of host gene exchange and recombination because RNA is encapsidated
No pre existing immunity against NDV reported
What is mean death time?
Mean death time is the time in hours for the minimum lethal concentration to kill all eggs in the dilution series
For example: 120 hours, MDT = 120 hours
What does MDT mean for virulence?
Less than 60 hrs: Velogenic
Between 60 and 90 hrs: Mesogenic
Greater than 90 hours: Lentogenic
What does ICPI mean for virulence?
Velogenic: >/1.5
Mesogenic: 1.5 - 0.7
Lentogenic: < 0,7
