RESEARCH ACTIVITIES ON NEWCASTLE DISEASE VIRUS: SEPRL · 2019. 7. 4. · PER08, EG11, TZ12 C C C...

David L. Suarez, DVM, PhD, ACVM

Research Leader Exotic and Emerging Avian Viral Diseases Research Unit

USDA- ARS, USNPRC, Southeast Poultry Research Laboratory

RESEARCH ACTIVITIES ON NEWCASTLE DISEASE VIRUS: SEPRL

NDV can infect over 250 bird species

Epidemiology and Adaptation• Class I APMV-1 viruses are found primarily in wild birds and

are non-virulent

• Class II viruses are found in wild birds and poultry

• Wild birds (waterfowl) have genotypes I, II, and X as endemic and non-virulent

• Poultry (chickens) have all genotypes. All genotypes except X have virulent (NDV) isolates. Genotypes I and II can be non-virulent or virulent. Other genotypes are virulent and are chicken adapted

• Pigeons (rock doves) have pigeon adapted virulent genotype VI

• Cormorants have cormorant adapted virulent genotype Va

• Spillover between poultry adapted to wild birds and wild bird adapted viruses to poultry occur frequently, but what is consequence?

What is the role of wild bird adapted NDV for poultry?

VaVI

Viruses from different genotypes

XIIIXIIVII

Va VI

PI13

Directly-inoculated birds, n=5 (0DPI)

Mortality, MDT, and clinical signs

Gross lesionsVirus Shedding

2, 4/5, 7DPI/DPPSerology - 14DPI (surviving birds)

Contacts birds, n=3

(2DPI)

CO10 PE08 EG11 TZ12

Wild bird viruses Chicken-adapted viruses

Low dose Medium dose High dose

CID50/ml

Chicken origin viruses

MDT- Medium dose

DI (DPI) Contact (DPP)PER08 4 5.7

EG11 4.8 7

TZ12 5 6.7

DPIPER08, EG11, TZ12

C

C

C

Ferreira et al, Veterinary Microbiology 235 (2019) 25–34

Virus shedding- Chicken viruses


Gross lesions- Chicken viruses

Chicken origin viruses

Virus DI (DPI) Contact (DPP)

PER08 4.2 6.3

EG11 7.4 8

TZ12 8.8 8.3

MDT- Low dose

PER08, EG11, TZ12DPI

B BB

Transmission pattern for chicken viruses

EG11 TZ12 PE08

Directly-

infected

(n=5)

2DPC 4DPC 7DPC 2DPC 4DPC 7DPC 2DPC 4DPC 7DPC

ND 6.1 NS 2.8 5.2 NS 2.8 6.3 NS

ND 2.0 5.7 ND ND 3.3 ND 7.3 NS

2.1 5.6 NS ND ND 2.9 ND 3.9 6.8

ND 5.6 NS ND ND 7.0 ND 7.4 NS

ND ND 6.7 ND ND 5.4 ND 3.4 6.2

Mean 2.1 4.8 6.2 2.8 5.2 4.7 2.8 5.7 6.5

Contacts

(n=3)

2DPP 5DPP 2DPP 5DPP 2DPP 5DPP

2.4 5.5 ND 5.9 2.9 7.2

1.9 5.4 ND 6.3 3.5 7.0

ND 5.0 ND 2.5 3.8 6.6

Mean 2.2 5.3 4.9 3.4 6.9

Low dose groups

CO10

PI13

DPIPE08, EG11, TZ12

B

A

C C B

Wild bird viruses

MDT- High dose

DI (DPI) Contact (DPP)PI13 NA NAC010 5.3 NA

CO10

PI13

DPIPE08, EG11, TZ12

B

A

CCC

MDT- Medium dose

DI (DPI) Contact (DPP)

PI13 NA NA

C010 7 NA

Clinical signs- CO10

Virus shedding – Wild bird viruses

Wild bird viruses- ICPI vs CID50/ml

Virus ID ICPI Log10 CID50/ml

PI13 1.20 >6.50

C010 1.55 5.97

PE08 1.78 <2.90

TZ12 1.88 3.05

EG11 1.88 3.65

Seroconversion wild bird viruses

Virus ID ICPIVirus dose**

(log10 EID50/ml)

DI-

HI titer (log2)

Contact birds

HI titer (log2)

PI13 1.2

2.1 <2 <2

4.1 3.5 <2

6.5 6.4 <2

CO10 1.55

2.7 <2 <2

4.3 <2 <2

6.3 8 <2

PI13: ROPI/USA/Allegheny/PA/ND0007199/2013

CO10: DCCO/USA/A00874288/650/2010

Conclusions

• Pigeon and cormorant lineage viruses required a high challenge dose to infect chickens

• Did not transmit efficiently to contact birds, even using a high infectious dose.

• Pigeon and cormorant lineage viruses are rarely found in poultry

• Both viruses had ICPI in chickens > 0.7 and a cleavage site compatible with virulent NDV

• Reduced virulence for chickens when the virus was given by a natural route of infection.


Pigeon NDV (PPMV-1) in chickens

Alexander and Parsons, 1986; Kommers et al., 2001; Kommers et al., 2002

Some experimental evidence that pigeon-adapted viruses can

gain virulence when passaged in chickens

California NDV Outbreak Main questions

• Phylogenetic relationships with other NDV

• Side-by-side comparison of related CA02, BE08 viruses

• Susceptibility of young and adult chickens to infection using different infectious dose

• Pathogenesis and shedding dynamics of the viruses and transmissibility to contact chickens

Can we apply all the knowledge obtained to facilitate risk assessment and control effort for the eradication of the CA02 virus?

Genotype V NDV Viruses

Dimitrov et al. 2019. Virology May;531:203-218.

Pathogenesis and transmission experiment with the California 2018, Belize 2008 and California 2002 NDV in

3-week-old SPF chickens

Swabs from DI at 1 to 4 DPI, and at

7DPITissues 4DPI

3 SPF contact birds at

2 DPI

All transmission birds swabbed at 2

and 5 DPP

Tissues (n=23): 1-3 DPI

CA18

High dose

Challenge 9 birds with:


CA02 BE08 CA18

Medium dose High dose

Mortality data 3-week-old SPF chickens


High dose

CA02

BE08

CA18

Medium dose

MDT (DPI or DPP)

Group DI BIRDS CONTACT BIRDS

Medium

CA02 5.2 7

BE08 6.4 7

CA18 4.6 6

High

CA02 4.2 5.7

BE08 5 7

CA18 4 5

Gross lesions in 3-week-old SPF chickens

CALIFORNIA 2018 CALIFORNIA 2002 BELIZE 2008congested organs after 3DPC

cervical subcutaneous edema

congested organs after 3DPC


congested organs after 3DPC


enlarged and mottled spleen

(atrophic in contact birds)

enlarged and mottled spleen mottled or pale spleen (atrophic

after 7DPC)

swollen kidneys with lobular

pattern

swollen (pale) kidneys with lobular

pattern

swollen kidneys with lobular

pattern

enlarged cecal tonsils, some with

hemorrhages

necrotic hemorrhages in ileum and

cecal tonsils

hemorrhages in proventriculus hemorrhages at the

proventriculus-gizzard junction

hemorrhages in proventriculus

greenish content in intestine hemorrhages and greenish content

in intestines

hemorrhages in larynx and upper

trachea

hemorrhages in larynx hemorrhages in larynx

enlarged thymus with

hemorrhages

flaccid thymus flaccid thymus, hemorrhages in

thymus

flaccid bursa swollen or atrophic bursa

eyelid hemorrhages eyelid hemorrhages eyelid hemorrhages

hemorrhages and necroses on

comb

hemorrhages on comb

Medium dose groups

RN

A v

iral

sh

edd

ing

(lo

g EI

D5

0/m

l)

High dose groups


Pathogenesis and transmission experiment with the California 2018, Belize 2008 and California 2002 NDV in

62- week-old SPF layer chickens

Swabs from DI at 1 to 4 DPI, and at

7DPITissues 4DPI

2 SPF contact birds at

2 DPI

All transmission birds swabbed at 2

and 5 DPP

Tissues (n=23): 1-3 DPI

CA18

High dose



CA02 BE08 CA18

Medium dose High dose

Mortality data SPF layers

MDT (DPI or DPP)

Group DI BIRDS CONTACT BIRDS

Medium

CA02 5.6 6.5

BE08 5.6 7

CA18 5.4 7

High

CA02 5.2 6

BE08 5.6 6.5

CA18 5.4 5.5

High dose

CA02

BE08

CA18

Medium dose


GROSS LESIONS IN SPF LAYERS

CALIFORNIA 2018 CALIFORNIA 2002 BELIZE 2008congested organs congested organs congested organs

enlarged and mottled spleen, pale

spleen, atrophic in one bird and contact

birds

pale spleen, mottled spleen enlarged and mottled spleen, atrophic

and pale in one bird 5DPC

swollen kidneys with lobular pattern swollen kidneys with lobular pattern swollen kidneys

hemorrhages in cecal tonsils

hemorrhages at proventriculus-

esophagus junction 2DPC, multifocal

hemorrhages in proventriculus 3DPC

hemorrhages at proventriculus-gizzard

junction

hemorrhages in proventriculus

yellow-brown foamy intestinal content

2DPC, green content 3 DPC,

hemorrhages 5DPC

Green content in intestines Green content in intestines

Hemorrhages in larynx Hemorrhages in larynx Hemorrhages in larynx

Hemorrhages in upper trachea Hemorrhages in upper trachea Hemorrhages in trachea

Small necroses in lungs

Hemorrhagic and necrotic yolk follicles Hemorrhagic yolk follicles Hemorrhagic yolk follicles

comb discoloration, cyanosis comb discoloration, cyanosis comb discoloration, cyanosis

eyelid hemorrhages eyelid hemorrhages eyelid hemorrhages

airsacculitis airsacculitis airsacculitis

flaccid liver with discoloration areas

hemorrhages in breast muscles

High dose groupsMedium dose groupsR

NA

viral sheddin

g (

log E

ID50/m

l)

Conclusions

• All three genotype Vb viruses had similar pathologies in both 3 week and adult birds

• NDV viruses are highly transmissible in chickens

• Because of similarities of viruses we can use data from previous research on CA02 viruses to predict CA18 viruses

• Pigeons can be infected but shed low amounts of virus

• Vaccination programs for broilers are poorly protective for virulent NDV challenge


Comparison MiNION vs MiSeq

2/3 Fusion (F) Complete genome

Samples from Mexico positive for NDV

SEPRL IDGenBank

numberSample Name

Bird

Specie

Near farms

(Jalisco, MX)

Collection

date

RRT-PCR (Ct

value)

1410 Ave1-01 sparrow A 08/11/17 12.41

1264* MK046916 Ave2-01 dove A 08/11/17 14.56

1265* MK046917 Ave3-01 pigeon A 08/11/17 13.29

1266* MK046918 Ave4-01 pigeon B 08/11/17 14.58

1267* MK046919 Ave5-01 pigeon B 08/11/17 14.08

1268 Ave6-01 pigeon B 08/11/17 14.24

1411 Ave7-01 chicken B 08/11/17 24.74

1270* MK046921 LV-7213 pigeon C 07/04/17 15.33

1412 LV-1517 pigeon C 07/04/17 25.99

1279 VX17144-2 pigeon B 10/10/17 14.25

1280* MK046920 LVVX17186-1 chicken C 10/10/17 14.51

Ferreira, Taylor et al. Virus Genes. 2019 Apr 12. doi: 10.1007/s11262-019-01663-1.

NDV in Mexico by MiNION sequencing

MinION

SEPRL

IDIsolate Name

NDV

Genotype% Identity

Consensus

length

NDV

reads

Mean

depth

1410 Ave1-01 Vc 97.9 1,285 3,303 1,808

1264 Ave2-01 Vc 98.2 1,254 15,651 12,320

1265 Ave3-01 Vc 97.9 1,285 2,072 1,301

1266 Ave4-01 Vc 97.9 1,285 9,082 5,567

1267 Ave5-01 Vc 98.1 1,285 39,709 26,349

1268 Ave6-01 Vc 97.9 1,285 21,030 13,898

1411 Ave7-01 Vc 97.9 1,285 3,576 2,679

1270 LV-7213 VIn 98.5 1,285 4,084 3,363

1412 LV-1517 VIn 97.6 1,222 6,196 5,317

1279 VX17144-2 VIn 98.4 1,285 267 242

1280 LVVX17186-1 Vc 97.7 1,285 4,197 3,078


NDV in Mexico by MiSeq sequencingMiSeq

MinION vs

MiSeq

SEPRL

ID Isolate NameNDV

genotype

%

Identity

Alignment

length

%

Coverage

Mean

depth

% Pairwise

Identity

1410 Ave1-01 ND NA

1264 Ave2-01 Vc 97.5 13,439 88.5 15 99.99

1265 Ave3-01 Vc 97.6 15,172 99.9 2,433 99.99

1266 Ave4-01 Vc 97.6 15,168 99.9 192 99.99

1267 Ave5-01 Vc 97.6 15,175 99.9 9,428 99.99

1268 Ave6-01 ND NA

1411 Ave7-01 ND NA

1270 LV-7213 VIn 98.1 15,168 99.9 1,135 99.99

1412 LV-1517 ND NA

1279 VX17144-2 ND NA

1280 LVVX17186-1 Vc 97.4 15,171 99.9 885 99.99Ferreira, Taylor et al. Virus Genes. 2019 Apr 12. doi: 10.1007/s11262-019-01663-1.

NDV in Mexico-MiNION


Conclusions

• The sub-genotype Vc continues to circulate locally only in Mexico• The presence of virus (genotype Vc) commonly isolated from

chickens in wild birds suggests spillover from poultry.

• First detection and complete genome of genotype VIn in pigeons from Mexico. • Our study demonstrates the epidemiological connection between

US and Mexican pigeon-adapted AAvV-1

• MinION could successfully be used for robust and rapid genotyping of NDV• Tree with same topology using 1220pb from MinION and complete

genomes from MiSeq

• Virol J. 2018 Nov 22;15(1):179. doi: 10.1186/s12985-018-1077-5.

https://www.ncbi.nlm.nih.gov/pubmed/30466441

Southeast Poultry Research Laboratory Exotic and Emerging Viral Disease

Thanks!!!• David Suarez Research Leader

• Claudio Afonso Lead scientist

• Mary Pantin-Jackwood

• Kiril Dimitrov

• Tonya Taylor

• Dana Goldenberg

• Iryna Goraichuk

• Catharine N. Welch

• Henri Kariithi

• Elizabeth Pusch

• Timothy L. Olivier

• Suzanne Deblois

• Dawn Williams-Coplin

• Diane Smith

• Ricky Zoller

• Roger Brock

• Keith Crawford

• Elizabeth Pusch

RESEARCH ACTIVITIES ON NEWCASTLE DISEASE VIRUS: SEPRL · 2019. 7. 4. · PER08, EG11, TZ12 C C C...

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