reverse genetics with animal viruses - vricentaur.vri.cz/news/prilohy/pril407.pdf · rinder pest...
TRANSCRIPT
Reverse Genetics with Animal Viruses
Teshome MebatsionIntervet, Research and Development
Viruses• DNA Viruses• RNA Viruses • Positive-strand
• Negative-strand
NSV-Human pathogensRabies HPIVMeasles HRSVEbola MarburgBorna Influenza
NSV-Animal pathogensRabies NDVCDV BRSVBPIV VSVRinder pest Influenza
Recovery of NNSV
Perspectives of Reverse Genetics
• Study of virus biology and virus host interaction
• Design of vectors to express foreign genes
• Developing improved attenuated vaccines
Rabies
Neurotropic fatal infection
Rabies
In developed countries
Rabies
In developing countries
• About 30,000 people die of rabies annually in India alone
• Nearly 96% of rabies is due to bites from dogs which are mostly stray and ownerless.
• Solution = Oral immunization of stray dogs in addition to routine vaccination
Genetically stable engineered rabies virus
(Step 1, attenuation)
The triple mutant ORA-D is genetically stable in suckling mice passages and in more than 25 cell culture passages in the absence of any Mab
ORA-D with Arg333 substitution is safe in adult mice
0
20
40
60
80
100
ORA-DSAD
102
108IC
Why is further attenuationof ORA-D required?
ORA-DSAD
20
40
60
80
100
0
IC
(Raux, et al., J. Virol. Nov. 2000), (Jacob, et al., J. Virol. Nov. 2000)
Dynein Light Chain (LC8)LC8 protein is involved in intracellular movement
Rabies P binds to LC8 (at AA 138-172)
LC8 is probably responsible for the axonal transport of rabies virus
Thus the goal of the second step of attenuation is to block this transport
Recombinant rabies viruses
SAD-L16
D333D - ∆P7D - ∆P11
L- ∆P7L- ∆P11
ORA-DP
LC8-P interaction Growth curve
A
B
L16L∆
7L∆11D333
D∆7D∆
11
N
P
LC8 3
4
5
6
7
8
9
4 18 24 48
Time (hours)
Tite
r (lo
g 10
ffu/m
l)L16L7L11D29D7D11
Pathogenicity of recombinant rabies viruses in mice
0
20
40
60
80
100
% M
orta
lity
Adult 2-day-old
Nude SCID
SAD-L16ORA-DORA-DP
Newcastle Disease Virus
Newcastle Disease Virus
• Causative agent of an economically important disease of poultry
• Controlled by post-hatch vaccination
Design and Developing Improved Attenuating Vaccines
• NDV In ovo vaccine• Marker NDV vaccine• NDV as a vaccine vector
Advantages of in ovovaccination
• Automated delivery ~30,000 eggs/hr
• Administration of a uniform dose
• Cost & labor saving• Reduces handling of
chickens• Immunity at the
earliest opportunity
Existing live vaccines
• Reduce hatchability• Further attenuation of vaccine strains
Determinant(s) of NDV virulence
• Efficiency of F protein cleavage• Velogenic• Mesogenic• Lentogenic
• Other virulence factors???
P-gene mRNA editing of NDV
NDV-P1 is attenuated for chicken embryos
0123456789
7 d. 8 d. 9-11-d.0
102030405060708090
100
7 d. 8 d. 9-11 d.NDV-P1rNDV
Titer Mortality
0
20
40
60
80
100
0 1 2 3 4 5 6Log10 EID50/ml
% M
orta
lity
NDV-P1 rNDV
V protein of NDV is a virulence factor
V protein of NDV is a Virulence Factor
• Mebatsion T, Verstegen S, De Vaan LT, Romer-Oberdorfer A, Schrier CC. A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos. J Virol. 2001 Jan;75(1):420-8.
• Huang Z, Krishnamurthy S, Panda A, Samal SK. Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist. J Virol. 2003 Aug;77(16):8676-85.
• Park MS, Garcia-Sastre A, Cros JF, Basler CF, Palese P. Newcastle disease virus V protein is a determinant of host range restriction. J Virol. 2003 Sep;77(17):9522-32.
NDV-P1- an embryo vaccine candidate
Hatch.Body Wt
0
20
40
60
80
100
P1 rNDV Control 01
2
3
4
5
6
3.5 4.3 5.4 Control
Mean HI titer
0
20
40
60
80
100
3.5 4.3 5.4 Control
% Survival
Marker NDV Vaccine
• A vaccine, in conjunction with a diagnostic test, that enables serological differentiation of vaccinated animals from infected animals.
• A virus lacking one or more nonessential, but immunogenic genes or immuno-dominant epitopes (IDE)
Highly immunogenic genes of NDV
Induce protective Abs
Induces non protective Ab
Recombinant NDV lacking an immunodominant epitope
anti-F anti-NP
1: wt NDV2: NDV-∆183: NDV-MHV14: NDV-MHV2
Cl.30
d18
NDV marker test
0
1
2
3
4
5
6
7
8
9
CONTROL CLONE 30INAC.
CLONE 30LIVE
D18 D18 +HVT/F
HI T
ITE
R
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
HI Titer Virion Peptide
OD
450 nm
Conclusions• Stepwise attenuated rabies virus as a safe and immunogenic
candidate vaccine for oral immunization of dogs
• Generation of an in ovo NDV vaccine candidate expressing low level of V protein
• Generation of a marker NDV candidate vaccine which enables serological differentiation between vaccinated and infected animals
• NDV can be designed to express foreign proteins for the development of broad spectrum NDV-based vector vaccines