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Evolutionary genetics of Newcastle disease virus
US Department of Agriculture, Agriculture Research Service, US National Poultry Research Center, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605, USA
Patti J. Miller, Kiril M. Dimitrov, Claudio L. Afonso
AVIAN FORUM ASIA
Chiba, JapanJuly 12-13, 2016
Newcastle Disease Team• Claudio Afonso, PhD: Lead ND Microbiologist• Kiril Dimitrov, DVM, PhD: Visiting Scientist from Bulgaria• Tim Olivier, MS: Biological Science Technician• Dawn Williams-Coplin: Biological Science Technician• Iryna Goraichuk, PhD: Post Doc from Ukraine• Poonam Sharma, PhD: Post Doc from France• Tonya Taylor, PhD: Post Doc from Clemson University• Stivalis Cardenas Garcia, DVM, PhD: Post Doc Univ. of Georgia• Valerie Marcano: PhD/DVM student UGA• Andrea Ayala, MS: PhD student UGA• Salmon Latif, DVM: PhD and Pathology Resident UGA• Mahmoud Sabra, DVM: Visiting Scientist from Egypt• Patti Miller, DVM, PhD: Veterinary Medical Officer• David Suarez, DVM, PhD: Research Leader Exotic & Emerging
Avian Viral Disease Research Unit
Athens, GA
Genotypes & Evolution
Introduction
Diagnostics & Vaccination
Newcastle Disease (ND)
Highly contagious viral disease of most (if not all) types of birds Reported in more than 236 species of birds representing 27
of 29 orders
Etiology: VIRULENT strains of Newcastle disease virus (NDV)
OIE*: Infections poultry with virulent NDV (vNDV)= ND
NDV of low virulence found world wide in wild birds and poultry species. Some are used as ND vaccines.
*OIE = Office International des Epizooties = World Organisation for Animal HealthThey coordinate, support and promote animal disease control; facilitate trade
1
JAN. to JUNE 2012
JULY to DEC. 2012
JAN. to JUNE 2013
Newcastle disease 2013-2014 WORLDWIDE68 countries reporting outbreaks in
domestic poultry
Jan 2012-June 2013
For comparison: HPAI & LPAI data for the same reporting time:17 countries reporting outbreaks in
domestic poultry Human Infection with mortality
ISRAEL- 58 (2012), 20 (2013)PAKISTAN- all months (2012), Jan- June (2013). INDONESIA-All administrative divisions (23): every month (Jan ‘12-June ’13)O
UTB
REA
KS
ND OUTBREAK DATA FROM OIE2
Newcastle disease: 61 countries reporting ND
outbreaks in domestic poultry
HPAI & LPAI: same reporting time:10 countries/HPAI 8 countries/LPAI 6 countries/both
ND OUTBREAK DATA FROM OIE 2014 Domestic PoultryJanuary –June 2014
July –December 2014
http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseasetimelineshttp://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseasedistributionmap
2015: countries reporting (26) ND & (23) HPAI domestic birds (as of April 2016)
2003
3
Glycoproteins: Fusion ( )&Hemagglutinin Neuraminidase ( )
Mast & Demeestere Diagnostic Pathology, Feb 2009
Newcastle disease virus
The immune system of the host is able to “see” the F & HN and make antibodies (Abs) to neutralize the virus.
HN/F bind & fuse to host cells. Neutralizing Abs inhibit attachment and fusion.
3’ Le NP P M F HN L Tr 5’
-Negative sense-Non-segmented-6 structural genes-pleomorphic-neutralizing Ab to HN & F~17 nm-Single-stranded-RNA
4
OIE:1) Intracerebral Pathogenicity Index (ICPI) > 0.7
2) > 3 multiple basic amino acids (R, K) positions113-116 of fusion cleavage site* & phenylalanine (F) @117
113 114 115 116 117
R Q R R F
K Q K R F
K R R R F
• *If cleavage site is not virulent, need to do ICPI• R= Arginine, K = Lysine
What are virulent NDV?5
NDV of low virulence (e.g. vaccines) with < 3 basic amino acids are cleaved by proteases that are found in the GIT & RT of the host, & therefore, the virus and lesions are mostly limited to these locations.
Virulent strains with > 3 basic amino acids in the F cleavage site are cleaved by proteases found throughout the body of the host, therefore, the virus is systemic.
NDV is activated when the Fusion protein is cleaved
Why is the cleavage site so important?
6
Genetic diversity of APMV-1 pre 2012
• APMV-1 comprise a highly diverse group of viruses
• All NDV isolates belong to one serotype (APMV-1)
• Several genetic groups (lineages/genotypes) have been identified
• Simultaneous evolutionary changes
• Lineages vs. genotypes • Late 1980s/early 1990s• Broad criteria• 2012, Diel• 2014, Consortium
Adapted from Cattoli et al., Vet Microbiol . 142, p168-176, 2012.
Genotype IV
Genotype III
Genotype V
Lineage 3Genotype VI
Lineage 4
Lineage 7
Genotype VII
Lineage 5
Genotype I
Lineage 1
Genotype II
Lineage 2
Unclassified
Lineage 6
LINEAGES ≠ GENOTYPE
7
Genotypes
class II: XVIII genotypes**some sub-genotypes
class I- one genotype
Virology 391, 2009, 64-72
• Mean Inter-population evolutionary distance between previous NDV genetic groups (containing at least 4!)
• Complete NDV genome • Complete NDV fusion Genotypes-average distances (nt)per site >10% w/bootstrap >60%Bootstrap- accuracy, confidence
Post 2012:Genotypes based on strict criteria
BOOTSTRAP is a computer-based technique for assessing the accuracy of almost any statistical estimate. Provides assessments of “confidence” for each clade of an observed tree, based on the proportion of bootstrap trees showing that same grouping.
8
Genetic distances rely on multiple sequence alignments
Number of base substitutions/site = averaging all sequence base pairs between aligned sequences with 1,662 nucleotides:[standard error estimates w/500 bootstrap replicates] & maximum composite likelihood model. SNOECK J. Clin. Microbiol. 51(7) 2013, 2250-60
Sub-genotypes: a) tree topology!, b) bootstrap > 60%, 3) mean evolutionary distances >0.03 & <0.1. (Infect., Gen., Evol. 12, 2012, 1770-79) & 4) specific residues (SNOECK, 2013)
J. Clin. Micorbiol. 51(7) 2013, 2250-60
Sub-genotypes: Ia, b; Va, b, c ; VIb, c, e, f, g; VIIb, d, e, f, g, h, i; XIIIa,b, XIVa, b; XVIIa, b; XVIIIa, b.
* Require multiple sequence alignments as input (replicates)
9
Distance is often defined as the fraction of mismatches at aligned positions, with gaps either ignored or counted as mismatches.
VII
VI,VII, VIII, IX,XII,XV
V
VII
VII
V, VI,VII
XVII, XVIII IX,
VIIi
XVI
VI, VII,XIII
XIV, XVII, XVIII
VIIi, XIII
VII
XIV, XVII XVIII
VIV,VI
XI
VII
II,III,IV
Courtesy of Dr. Stivalis Cardenas Garcia
More details – Infect. Genet. Evol. 2016 Apr; 39:22-34. doi:10.1016/j.meegid.2016.01.008
VI, VIIiVIIi
VI
XII
VIIIVII
V
V
V
11
Four defined ND panzootics1.1926 Indonesia/England; genotypes II, III, IV (now rare)
2.1963-1970s Middle East/Europe/Americas; genotype V; first associated w/psittacines, now poultry. USA 2002, cormorants Canada/USA 2008-2014, Mexico 2013. Rare outside of Americas; Croatia, Slovenia, Bulgaria, Korea
3.1960s “variant NDV” from pigeons N. Africa & Middle East; 1980s ~worldwide and ongoing. Can infect poultry species.
4.1980s Asia; genotype VII Africa, Western Europe, South America (2008); chickens, geese, ducks; ongoing
5.? 2014 reported possible 5th panzootic started 2010 Indonesia Israel/Pakistan (Turkey/Bulgaria); chickens, pheasants, parrot; genotype VIIi; ongoing
12
NDV Genotypes over decades-using GenBankcomplete fusion genesII
VI
VII
XVII
XIII
XII
V
Dr. Claudio Afonso
13Virus Genes (2014) 49:89–99 DOI 10.1007/s11262-014-1075-7 Characterization of complete genome sequence of genotype VI and VII velogenic NDV from Japan. Dennis V. Umali, Hiroshi Ito, Kazutoshi Shirota, Hiromitsu Katoh, Toshihiro Ito. Chickens isolates 1969 VIc, 1987 VIc, 1999 VIIe
Poultry Sci (2015) 94 (5) 890-897. Atypical velogenic Newcastle disease in a commercial layer flock in JapanDennis V. Umali, Hiroshi Ito,Kazutoshi Shirota,Toshihiro Ito,Hiromitsu Katoh. Chicken/2002 VIId
NDV IS EVOLVING – BUT HOW FAST?Herczeg et al. (2001) & Czeglédi et al. (2002) indicated that the expected nucleotide
sequence change of NDV caused by natural evolution is estimated to be
approximately 1% per decade (or 0.1% per year).Herczeg et al., 2001. Avian Pathol. 30:163-168.
Czeglédi et al., 2002. Epidemiol Infect. 129:679-688.
Fusion gene
Complete genome
Fusion gene (wild birds)
Miller et al., 2009 (Virology, 391:64-72)
Ramey et al., 2013 (Arch Virol. 158:2495-2503)
Courtesy of Dr. Kiril Dimitrov
14
Virulent genotype II
& IX look to be
recent
introductions, not
undergoing
natural
evolution.
Fusion gene substitution rate estimated using relaxed lognormal molecular clock
Genotype II virulent strains
Genotype IX virulent strains
Summary statistics
Mean7.05 x 10-5 2.05 x 10-5
Standard error3.48 x 10-6 6.41 x 10-7
Median5.37 x 10-5 1.51 x 10-5
Effective sample size361 992
Important to selectSequences from GenBank carefully When doing analysis on Rate of change.
15
Dimitrov, J Clin Micro, 2016
Does vaccination affect evolution?
Intensive NDV vaccination program
began in 2002
Belize ND outbreak 2008 more related to older NDV
16
Virulent NDV genotype VII- genetic drift & spread
• F. Perozo 1st
reported VII Venezuela in 2012 from 2008 outbreak
• In Columbia as early as 2006
• In both countries as recent as 2014 and 2015
Dr. Afonso
17
Dr. Afonso
Peru/2008 outbreak-reported 2012 closely related to VII-more isolates China revealed new genotype XII-now know XII was in Peru as early as 2004 from GenBank submissions in January, 2016-as early as 2009 spread to Columbia
18
Implications of NDV Evolutionary Dynamics
100
7096909092
10092
94
96 9498 88
78
100
86100
98
94
96
86
100
8888 100
100
100
7888
98
10078 86 100
84100
86
96
100
98 84100
96
100 100
100
86
72
100
100
100
100
88
96
100
94
10078100100
100
98
100
84
100100
86
100
86100
98
98
100
96
100
9272
96
100
100
100
10086 10
0
98 100
100
90
90
VII
V
VI
III
III/IV
XI
IX
• Diagnostic failures and difficulties for disease control
New diagnostic tests needed
19
Diagnostic tests• Antibodies - past infection
• Without worldwide vaccination cannot tell infected from vaccinated
• Virus Isolation- SPF
• Grow virus, HA+, RNA, sequence, rapid assay
• Rapid diagnostic tests
• RT- PCR
• Real time RT-PCR
• Florigenic probes using Taqman polymerase
• Monitor accumulation amplicon in real time
• Also use SyBr-green
• Fast results
20
Matrix Gene Real Time RT-PCR Assay
• Conserved region needed- made to detect all (most) APMV1
• Matrix gene
• Allows rapid detection from single RNA preparation
• But…does not detect class I viruses (approximately 70% of viruses of wild birds) or a virulent NDV from Pakistan Mukteswar (class II genotype III from 1940s)
J Clin Microbiol. 2007 Apr;45(4):1310-4 and May 2010,48 (5):1892–94
21
Genotype V(California)
Vaccines
Class I
The Matrix Test:
Relationship with Phylogeny
This is not surprising!
22
SEPRL L Gene Primer/Probe Development (Multiplex)
``````````````````````
Conserved region =
Polymerase gene
• Alignment from 37
sequences
• Software used for initial
selection
• Hand selection of a variety of
primers with a single probe
23
NDV Polymerase Matrix
Isolate # Site Year Class FAM Cycles FAM Cycles
AGWT -98-005 AK 1998 I POS 17.69 POS 30.81
MC-110 FR 1977 I POS 20.99 SUSP 36.18
380723 -2 MA 2005 I POS 15.95 POS 34.46
375204 -1 NY 2005 I POS 15.19 SUSP 35.36
375459 -4 NJ 2005 I POS 15.86 POS 34.6
371831 -1 NY 2005 I POS 14.34 SUSP 35.56
373225 -4 NY 2005 I POS 15.48 SUSP 35.92
37219 5-1 RI 2005 I POS 14.9 POS 34.63
377813 -34 NJ 2005 I POS 15.53 - 0
376880 -4 NY 2005 I POS 16.07 - 0
381913 -5 NJ 2005 I POS 15.8 - 0
3813 22-7 NY 2005 I POS 16.11 - 0
378725 -2 NJ 2005 I POS 14.66 - 0
378723 -4 NJ 2005 I POS 15.75 - 0
378722 -1 NJ 2005 I POS 15.7 - 0
372414 -10 NY 2005 I POS 15.27 SUSP 38.92
373356 -2 NJ 2005 I POS 15.44 - 0
1368.3 HK 2005 I POS 19.44 - 0
1513.16 HK 2005 I POS 17.64 - 0
1525.16 HK 2005 I POS 16.52 - 0
1875.16 -17 HK 2005 I POS 19.11 - 0
1939.4 HK 2005 I POS 14.69 - 0
USA/B1-47 US 1947 II NEG 0 POS 21.97
PT-87-164 OH 1987 II POS 29.59 POS 12.34
MLD-86-ca5 OH 1986 II POS 29.48 POS 14.2
SEPRL Polymerase Test Can Detect Class I NDV 24
The Fusion RT-PCR Test to Detect Virulent NDV
Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples
Journal of Clinical Microbiology, January 2004,
The problem:
It does not detect Dove/Italy/2000
25
2001
-AY
4717
32
0.02
Dove Italy was not alone!
A Large
group of
pigeon NDVs
isolated
worldwide
had very
similar
sequences at
the Fusion
cleavage site
26
Detection with Pigeon-specific Probes (SEPRL)
Using the Fusion Protein Real time RT-PCR primers
Accession Isolate description MatrixStandard
FusionFusion Pigeon-
specific
EU477193 Eurasian Collared Dove/US/TX2334/2003 16.83 0 23.55
EU477194 Eurasian Collared Dove/US/TX3817/2004 18.03 0 24.66
EU477195 Eurasian Collared Dove/US/TX6295/2006 16.71 0 23.55
EU477196 Eurasian Collared Dove/US/TX6306/2007 15.36 0 21.63
EU477197 Eurasian Collared Dove/US/TX6338/2007 17.33 0 24.59
EU477188 Dove/US/TX-B2580/2004 16.53 0 22.3
EU477189 Pigeon/US/RI166/2000 18.32 0 19.97
EU477198 Pigeon/US/TX3377/2004 16.22 0 21.45
EU477190 Pigeon/US/TX3503/2004 19.24 0 25.77
EU477199 Eurasian Collared Dove/US/TX3908/2004 18.6 0 27.26
EU477191 Eurasian Collared Dove/US/TX3988/2004 17.79 0 24.44
EU477200 Mourning Dove/US/TX4048/2004 17.81 0 23.46
EU477201 Eurasian Collared Dove/US/TX4078/2004 17.07 0 24.33
EU477202 Pigeon/US/TX4142/2005 17.66 0 27.54
EU477192 Eurasian Collared Dove/US/TX4156/2005 17.23 0 22.78
J Vet Diagn Invest. 2006 Nov;18(6):519-28
and J Clin Microbiol. 2008 Oct;46(10):3303-10.
27
The September 10, 2008 Outbreak in Cormorants from Five Minnesota lakes were confirmed to have virulent Newcastle disease, according to the Minnesota Department of Natural Resources (DNR). 2010 seagulls and cormorants in Maryland, 60 miles from Delmarva (Poultry Industry) were also infected with vNDV.
Real Time PCR Detection (the Fusion Assay fails)
Isolate Species Yr Matrix Fusion Sequence02US072 Game Fowl 2002 22.21 24.91 TGAGGAAGGAGACAGAAACGC
CC02 Cormorant 2002 23.19 30.78 TC..............................................T
19529 Cormorant 2008 31.08 0 TA..............A..............................T
442 Cormorant 2008 22.08 0 TA............RA..............................T
• Fusion Assay failed for genotype V vNDV• All together 6 cormorant isolates were negative on real time rt-pcr for fusion• Importance of VI in SPF embryonating chicken eggs (embryo death) especially lack of HA• Since 2002 cormorant vNDV don’t HA
28
cormorant
• Molecular assays that rely on genome sequence must be tested will all new NDV strains to ensure the assays are still valid
• Assays may (will eventually?) need to be modified –either primer(s) or probe or you can create a multiplex to detect a broad range of strains
• This could also affect your sequence results if you are using NDV specific primers
29
NEX GEN Sequencing
• Sequence hundreds of millions of short (35bp-100bp) sequences in a single run!
• Few common platforms: 454, Solexa/Illumina or SOLiD (ABI)• ~$1,500 one human genome in a week. • Problem: the amount of data is enormous• Bioinformatics necessary• Detect many avian viruses or bacteria from one sample• 30 samples in one run• Run not just with RNA extracted from NDV infected fluids, but also working
on sequencing directly from extracted swabs and from formalin fixed tissue samples
30
NDV Vaccines & Evolution• B1 & LaSota~ live and inactivated
vaccines are universally used in the poultry industry in the U.S.
• These current ND vaccine strains are phylogenetically similar to the NDV strains isolated in the 1940s-50s (class II, genotype II)
• They differ phylogenetically from recent outbreak viruses of ND isolated in U.S that are related to the virulent Mexican strains (class II, genotype V)
• Still all NDV belong to one serotype
31
How to control ND?
1. BIOSECURITY IS KEY-Shower into chicken facilities-Change shoes and clothes-Move from cleanest to “dirtiest” birds-Keep out wild birds-Secure sources of water, feed, vaccines-Do not spread ND infected litter
32
How to control ND?2. VACCINATION
While we may still see egg drops in well-
vaccinated layers, & we still have to
administer booster vaccinations every 3-4
weeks in areas with vNDV, for the most part
we can control morbidity & mortality.
33
e.g. For NDV strain CA/USA/2002, a non-vaccinated 5 week broiler might only require 10,000 virus particles to become infected. That same broiler vaccinated & with an HI antibody titer of 64 may now need 1,000,000 virus particles to be infected.
* Well-vaccinated birds still become infected even if they do not have clinical signs of disease*
Vaccination Goal #1: Increase the amount of vNDV vaccinated birds need to receive to become infected
NDV found later incidentally in a flock infected with IBV. Flock was well vaccinated for NDV –no signs of disease.
34
Vaccination Goal #2: Decrease the amount of vNDV vaccinated birds will shed after being infected at least 100-fold (e.g. shedding < 10,000 virions instead of 1,000,000 in 1ml of saliva).
ORAL SHEDDING-4 d PI with CA PREVENT MOREBIRDS FROM RECEIVINGTHE DOSE NEEDED TO BE INFECTED
35
ConclusionsNDV will continue to evolve. Diagnostic assays must always be tested and re-tested with new NDV isolates.
Biosecurity is critical no matter what vaccine you use to allow birds enough time to develop a proper immune response. No vaccine will work if immunosuppressive agents are present as co-infections.
Careful with cold chain, preparation & administration of vaccines, especially when using spray or water. It is critical to use a proper dose and to check backtiters of vaccines. >85% sero-conversion for herd immunity is necessary.
36
Acknowledgements• Merial
• David Suarez and Claudio Afonso
• NDV Team
• Animal Caretakers• Bill Gagnon• Ronald Graham• Keith Crawford• Roger Brock• James Doster
Thank you!
QUESTIONS?