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Merial Avian Forum 2014
Paris
25 April 2014
Richard Irvine BVetMed, PGCertILHP, MSc(CIDA), Dipl.ECPVS, MRCVS
Head of National Reference Laboratory (UK) for Statutory & Non‐statutory Viral Diseases of Poultry
Deputy Head EU/OIE/FAO International Reference Laboratory for AI & ND
AHVLA‐Weybridge
Richard.Irvine@ahvla.gsi.gov.uk
 EU/OIE/FAO International Reference Laboratory for Avian Influenza (AI) and Newcastle Disease (ND)
 OIE Reference Laboratory for Swine Influenza  Disease and scientific consultancy for AI/ND/Swine Influenza and other viral diseases of poultry: aiwrl@ahvla.gsi.gov.uk
Microorganisms’ variability:
Epidemiological consequences
Introduction
• Diseases of importance in poultry
– RNA viruses
• Biology & mechanisms
– Replication, mutation, selection
• Epidemiological consequences – Viral variability: R&D, Diagnosis, Prevention & control
Microorganisms’ variability
• Different microorganisms can infect and cause disease in poultry, and their properties may change over time
• Non‐infectious causes of bird and flock production problems are also very important:
– Flock management & husbandry
– Feed & water
– Environment & hygiene
Some pathogens & diseases of importance
• Bacterial
Infectious Coryza, Fowl cholera, E.coli, Staphylococcus aureus, Salmonellosis, Mycoplasmosis, Enterococcus hirae, Erysipelas, ORT, Clostridium perfringens, Riemerella anatipestifer
• Protozoal
Coccidiosis, Blackhead, Spironucleosis
• Fungal
Aspergillosis, Mycotoxicosis, Candidiasis
• Viral
Avian Influenza, Newcastle disease, IBV, Gumboro, TVP, HEV, Reoviral tenosynovitis, aMPV (TRT), Rotavirus, DVH I/II, CAV, Marek’s disease, ILT, Leukosis, Pox, IBH, EDS’76, MSD, DVE, GPV
• Parasitic
Red mite, Helminth worms
Some viral diseases of importance in poultry
• RNA viruses
Avian Influenza, Newcastle disease, IBV, Gumboro, aMPV (TRT), Reoviral tenosynovitis, CAV, TVP, HEV, BLSDV, Rotavirus, DVH I/II
• DNA viruses
Marek’s disease, ILT, Leukosis, Pox, IBH, GE EDS’76, MSD, DVE, GPV
Diseases of health, welfare, financial and economic importance to both poultry producers and the industry
RNA viral diseases of importance in poultry
• RNA viruses
Avian Influenza, Newcastle disease, IBV, Gumboro, aMPV (TRT), Reoviral tenosynovitis, CAV, TVP, HEV, BLSDV, Rotavirus, DVH I/II
• The nucleic acid that makes up the viral genome is RNA • Successful viral infection of host cells by RNA viruses requires many steps, including viral RNA replication and protein synthesis
• Replication requires an RNA‐dependent RNA polymerase enzyme
Viral RNA replication & synthesis +
Viral protein synthesis
↓
New progeny viruses
Mutations are introduced during viral RNA synthesis by the insertion of incorrect nucleotides by RNA polymerases into the new viral genome that is being made
RNA viruses are prone to error & mutation
• RNA viruses mutate at a higher rate than DNA viruses:
o DNA viruses make ~1 error per 107‐109 nucleotide copied
o RNA viruses make ~1 error per 103‐105 nucleotide copied
• This high rate of mutation comes from the lack of proof‐reading ability in RNA polymerases during the viral replication process o DNA viruses also make mistakes, but they can correct them
• RNA viruses have low copy fidelity and hence are error‐prone
o Each new RNA virus produced is likely to have one or more mutations RNA viral diversity & evolution
• Large numbers of new progeny viruses are produced during each round of viral replication
• Each round of viral replication therefore produces thousands of co‐existing mutants
• Viral population ‐ swarm ‐ of quasispecies
• Mutations may provide a selective advantage or disadvantage – ‘viral fitness’
Lauring & Andino (2010). Quasispecies Theory and the Behavior of RNA Viruses.
PLoS Pathog 6(7): e1001005. doi:10.1371/journal.ppat.1001005
RNA viral quasi‐species & ‘fitness’
• Genetically distinct, but functionally linked, virus variants (genotypes) will co‐exist in an infected host, eg. a chicken
• Some genotypic variants (viral quasispecies) may be ‘fitter’ than others: o RNA mutation that result in a selective advantage for the virus
o Immune escape mutants; transmissibility; tissue tropism, etc.
• Genotypic diversity results in different viral phenotypes and therefore influences the pathogenesis of viral infection in the host
Mechanisms for RNA viral diversity
• RNA viruses
Avian Influenza, Newcastle disease, IBV, Gumboro, aMPV (TRT), Reoviral tenosynovitis, CAV, TVP, HEV, BLSDV, Rotavirus, DVH I/II
• Genetic drift
Point mutations – nucleotide insertions
• Genetic shift
Re‐assortment/recombination of genes
Generation of novel genotypes and/or phenotypes eg. New strains (AI, ND, IBV, IBDV); LPAI to HPAI; APMV‐1 to NDV RNA virus variability Epidemiological consequences
Implications of viral variability: Diagnosis & control
1. Altered virulence & pathogenesis
– Differences in the development and severity of pathology & clinical signs
– R&D to understand implications for host, and detection approaches
2. Changes in antigenicity & immunogenicity
– Virus: ‘Escape’ host immune responses
– Host: Degree of vaccine protection vs. breaks in face of viral challenge 3. Challenges for diagnostic laboratory methods
– Use of conserved regions for diagnostic tools, eg. ELISA, PCR
– Disease criteria & definitions: internationally‐recognised standards
Two mechanisms for genetic changes
• Drift through accumulating point mutations in a single gene
– Influenza: HA has highest rates
• Shift by reassortment and/or recombination
– Exchange of genes/segments
• Examples: AI, ND, IBV
Li et al, (2004). Genesis of HPAI in East Asia.
Nature, 430(6996), pp209‐213.
Influenza A Virus
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Family Orthomyxoviridae
Genus Influenzavirus type A
Segmented RNA virus
Subtypes based on surface ‘spike’
glycoproteins
– Heamagglutinin (HA): H1-H16*
1. Antigenic, immunogenic
2. Host cell receptor binding
– Neuraminidase (NA): N1-N9*
1. Virion release from host cells
*in birds
Reassortment
Two influenza viruses infect the same cell
H1N1
H2N2
Plus 252 other combinations
H1N1
H2N1
H1N2
H2N2
Dennis Alexander
Two Avian influenza pathotypes
• LPAI
• HPAI
INFLUENZA VIRUS PARTICLES
Dennis Alexander
Classification based on disease in chickens
HA cleavage site sequence determines pathotype
R/KET
Single basic amino acid (R or K)
Multiple basic amino acids
Cleavage site point mutation(s) result in switch from LPAI to HPAI phenotype & genotype, after LPAI virus introduction to poultry from wild birds. H5/H7 AIV only.
OIE & EU definitions of Notifiable AI
Infection of poultry or other captive birds caused by any influenza A virus of the subtypes H5 or H7 is reportable.
Classified as HPAI if:
(i)An intravenous pathogenicity index (IVPI) in 6‐week‐old chickens >1.2 (max. 3.0)
(ii)H5 or H7 with multiple basic amino acids at the cleavage site of the HA protein… cleaved by a ubiquitous host protease
Confirmed disease requires immediate implementation of control measures
Newcastle disease virus
• Family Paramyxoviridae, Genus Avulavirus
• Avian paramyxovirus serotype 1 (APMV‐1)
• Virulent (NDV) and avirulent strains based on Fusion (F) gene cleavage site sequence
• RNA genome
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–
–
–
~15kb negative sense, single stranded non‐segmented
6 genes (8 proteins)
ND Fusion gene: Virulence & Clinical signs
Avirulent Virus
Virulent virus
Limited tissue/protease distribution
Mild or asymptomatic infection
Unlimited tissue/protease distribution
Severe, systemic disease & death
Typical avirulent motif: XRQXR * L
Virulent motifs: RRQRR * F / XRQRR * F
3'
55
1746
1451
1241
1792
2031
6704
NP
P
M
F
HN
L
2
1
1
35
47
5'
55
Phylogenetic data & viral relationships
• Study the evolutionary relatedness between viruses using genome sequencing data
• Particularly useful for rapidly evolving viruses
• Virus classification (clades)
• Understand epidemiology (source and spread)
• Inform prevention and control policies
Modified from: who.int/csr/disease/avian_influenza/H5N1evoconceptualdiagram.pdf
Differences in H5 AI viruses between Western & Eastern hemispheres
Eurasian H5N1 HPAI
Eurasian: Other H5 AIVs, mainly LPAI
American H5 AIVs
PCR detection of AI viruses: Matrix gene
• Matrix (M) gene highly conserved across all AI virus subtypes (H1 – H16), and across all geographic regions
• Continued evolution of AI viruses eg. Eurasian lineage H5N1 HPAI
• Mismatches with reverse primer (SEP2) of Matrix gene RRT‐PCR
Match to Clade
SEP2 sequence (Spackman et al 2002)
2.2
CAGAAACTTGAAGATGTCTTTGCA
A/muscovy duck/Vietnam/F23/09
2.3.2
CAGAAACTTGAGGATGTCTTTGCA A/chicken/Romania/10580/10 2.3.4
CAGAAACTTGAGGATGTCTTTGCA H5N1 HPAI isolate
A/chicken/Nepal/5185/10 CAGAGACTTGAAGATGTTTTTGCA
M gene RRT‐PCR
M1 ORF, segment 7
5’
Spackman et al (2002); 101 bp
M1
3’
Nagy et al (2010); 182 bp
Standardisation of robustly validated diagnostic methods in OIE & EU Manuals supported by laboratory ring trials.
Slomka et al., (2012). Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam, Avian Pathology, 41,(2), pp177‐193.
M gene (1)
M gene (2)
H5 HA2
Suspect AI/ND case in poultry flock
Official veterinary visit
Samples received in lab
≤ 12 hours: M gene + H5/H7 NAI or APMV‐1 PCR positive
≤ 36 hours: Virulent sequence
48 hours: Isolation of AI or ND virus
72 hours: Virus subtyping completed
Day 7: Virus isolation complete
Day 14: IVPI or ICPI complete
Infectious Bronchitis Virus (IBV)
• Global distribution of many different IBV serotypes
– Variation within and between countries and regions
– Indigenous IBV variants in many regions
• IBV is of major economic, health and welfare importance to poultry & poultry industry worldwide
• Continued emergence and evolutionary divergence of IBV variants
– Changes in Spike (S) protein amino acids – Novel variants do not always persist & spread, but some do, causing major disease and control challenges
Global distribution of IBV, 1950 onwards
IBV Serotypes identified
Europe
Asia
Americas
Australia
Mass
D207
D212
D3128
D3896
D1466
PL‐64084‐France
A224.74/Italy
B1648‐Belgium
UK/918/67
UK6/82
UK/142/86
UK793/B
624/Italy
QX (D388)
Mass
Conn
Gray
Ark 99
N1/52‐T
Kb8523‐Japan
793/B‐India
NRZ‐China
HV‐China
SAIB3‐China
KM91‐Korea
EJ95‐Korea
A1121‐Taiwan
QX
Mass
Conn
Florida
Clark 333
Ark 99
SE17
JMK
Iowa 97
Iowa 609
Holte
Gray
Main 209
DE/072/92
Chile 14
50/96‐Brazil
22/97‐Honduras
A‐Vac1
B‐Vic S
C‐N1/62
D‐N9/74
E‐01/73
F‐V2/71
G‐V1/71
H‐N1/75
I‐N2/75
J‐N3/63
K‐T1/82
L‐N1/88
M‐03/88
0‐NT2/89
P‐N1/81
Q‐V18/91
Adapted from: Ignjatovic, J. & Sapats, S. (2000) Avian Infectious Bronchitis virus. Rev. Sci. Tech. Off. int. Epiz.,19 2, pp. 493‐
508. Data also from: de Wit et al (2010) Infectious bronchitis virus in Asia, Africa, Australia and Latin America ‐ history,
current situation and control measures. Rev. Bras. Cienc. Avic. 12(2),pp.97‐106; de Wit et al (2011) Infectious bronchitis
virus variants: a review of the history, current situation and control measures. Avian Pathology, 40(3),pp.223‐235;
Jackwood, M. (2012) Review of Infectious Bronchitis Around the World. Avian Diseases, 56,pp.634‐641.
The emergence and evolution of IBV variants can occur by:
‐Mutation
‐Recombination Shu‐Ming et al., (2013) Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA‐binding activity. Vet Micro, 162(2), pp.408‐418. S1 gene sequencing & phylogenetic data reveal IBV diversity
de Wit et al., (2011) Infectious bronchitis virus variants: a review of the history, current situation and control measures. Avian Pathology, 40(3),pp.223‐235
IBV variants have different properties
Mutation Recombination IBV variants
Variations in phenotype & genotype
Virus Antigenicity
& host Vaccinal interactions responses
Virulence
Tissue tropism
Clinical signs
Pathology
After: Montassier, H.J. (2010) Molecular Epidemiology and Evolution of Avian Infectious Bronchitis Virus. Rev. Bras. Cienc. Avic. [online] 12(2)pp.87‐96
False layer syndrome
Enlarged kidneys due to infection with a nephropathogenic IBV strain
Images: AHVLA
Vaccines are an important tool
• Live attenuated vaccines eg. IBV, IBDV
– Good protection if ‘match’ with same serotype in‐region – Variable/poor protection if infection with heterologous variant (or other causes of disease!)
• Use of two different serotypes of live IBV vaccine (eg. Mass + variant) can provide broader protection and efficacy – Not always need a new vaccine; ‘Double live vaccine’ schedules
– Live prime/inactivated boost schedules for breeders & layers
• However, may not protect against all virus variants
Cross‐protection between IBV strains varies with S1 gene homology
de Wit et al., (2011) Infectious bronchitis virus variants: a review of the history, current situation and control measures. Avian Pathology, 40(3),pp.223‐235
Factors to consider: Vaccination & diversity
• Vaccination
– Must be properly administered
– Can reduce the severity of clinical signs and mortality
– Has best effect if there is a good antigenic match between vaccine and field virus: are new vaccines always needed?
• Surveillance in vaccinated populations
– Purpose? Virological vs. serological monitoring
– Serology tests often not differentiate field and vaccine strains as based on conserved epitopes (unless DIVA strategy)
Implications of viral variability: Diagnosis & control
1. Altered virulence & pathogenesis
– Differences in the development and severity of pathology & clinical signs
– R&D to understand implications for host, and detection approaches
2. Changes in antigenicity & immunogenicity
– Virus: ‘Escape’ host immune responses
– Host: Degree of vaccine protection vs. breaks in face of viral challenge 3. Challenges for diagnostic laboratory methods
– Use of conserved regions for diagnostic tools, eg. ELISA, PCR
– Disease criteria & definitions: internationally‐recognised standards
Prevention & Control
• Prompt veterinary investigation of flocks
• Consider differential diagnoses and correct sampling • Robustly validated, appropriate & timely lab testing
• Surveillance and R&D to understand viral diversity & implications
• Information to support disease prevention & control decision‐making
– Understanding epidemiology , risk pathways and control options
– Appropriate vaccine selection (+ correct handling, storage, administration)
• Biosecurity is good for your business
Prevention & Control
The most efficient and effective method for the control of infectious diseases in poultry is to prevent infection from entering the country, area, farm or flock
Acknowledgements
• Avian Virology & Mammalian Influenza Group, AHVLA Weybridge – Ian Brown, Dennis Alexander, Ruth Manvell, Jill Banks, Marek Slomka, Wendy Howard, Sharon Brookes, Brandon Londt, Scott Reid & teams
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Poultry industry, owners & keepers
Private Veterinary Surgeons
National & International laboratories & field staff
Defra
EU
OIE
FAO
Merial
Thank you for your attention
Richard.Irvine@ahvla.gsi.gov.uk