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

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ORA-DSAD

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108IC

Why is further attenuationof ORA-D required?

ORA-DSAD

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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

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4 18 24 48

Time (hours)

Tite

r (lo

g 10

ffu/m

l)L16L7L11D29D7D11

Pathogenicity of recombinant rabies viruses in mice

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% 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

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7 d. 8 d. 9-11-d.0

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7 d. 8 d. 9-11 d.NDV-P1rNDV

Titer Mortality

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% M

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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

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P1 rNDV Control 01

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3.5 4.3 5.4 Control

Mean HI titer

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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

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CONTROL CLONE 30INAC.

CLONE 30LIVE

D18 D18 +HVT/F

HI T

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R

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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