Presence of Virulent Newcastle Disease Virus in ...

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Feb 18, 2015 - AY288998/cockatoo/Indonesia/14698/1990 Ancestral. JN986837/chicken/NL/152608/1993 Ancestral. AY444497/moluccan/Indonesia/904/87 ...
JCM Accepted Manuscript Posted Online 18 February 2015 J. Clin. Microbiol. doi:10.1128/JCM.02818-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Short-Form Paper

Title

Presence of Virulent Newcastle Disease Virus in Vaccinated Chickens Farms In Pakistan

Abstract

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One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens in multiple farms despite of the existence of high average NDV specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.

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Authors: Shafqat Fatima Rehmania, Abdul Wajida,b, Tasra Bibia, Bushra Nazira, Nadia Mukhtara, Abid

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Hussainc, Nazir Ahmad Loned, Tahir Yaquba,b, Claudio L. Afonsod

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a

Quality Operations Laboratory (QOL), University of Veterinary and Animal Sciences, Lahore 54000, Pakistan. b Institute of Biochemistry and Biotechnology (IBBt), University of Veterinary and Animal Sciences Lahore 54000, Pakistan. c Poultry Disease Diagnostic lab, Gakkhar, Gujranwala, Punjab, Pakistan. d Exotic and Emerging Avian Disease Research Unit, Southeast Poultry Research Laboratory, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA.USA. Key Words: Newcastle disease (ND), reservoir, poultry, vaccination Running title: Virulent NDV in vaccinated chickens

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Background. Newcastle disease (ND) is one of the most devastating diseases affecting the

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poultry industry worldwide (1). It is caused by avian paramyxoviruses of serotype 1 (APMV-

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1), also known as Newcastle disease virus (NDV) (1). NDV may be classified into classes

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and subdivided into up to 18 genotypes based on genetic diversity (2). Currently pigeons and

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in cormorants are two well recognized reservoirs of virulent NDV (vNDV) (3, 4), however

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those species normally carry viruses of genotypes Va and VI (1) and the reservoir for other

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highly virulent viruses that commonly circulate in poultry (genotypes Vb, VII, and XII to

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XVIII) is unknown.

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The failure to control ND with vaccination alone in endemic countries underscores the

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importance of understanding mechanisms of vNDV maintenance. It has been shown that

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under experimental conditions vaccines do not prevent replication of highly virulent viruses,

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thus circumstantial evidence suggests that the capacity of vaccinated birds to shed viruses

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may allow vaccinated poultry to act as reservoir (1). As culling is the preferred control

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strategy in the developed world and the epidemiology of vNDV in endemic countries is not

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well understood, evidence supporting a role of vaccinated chickens as reservoir is missing.

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Experimental Details. Three hundred tracheal samples from sick or dead birds and 207

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blood samples from live vaccinated chickens in production were analyzed for the presence of

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vNDV during a 3 months period (March to May, 2013) after a serious outbreak in Pakistan

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(2011-2012) was contained (10). Serum samples were collected mostly at three weeks of age

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from day one vaccinated chickens that were not showing clinical symptoms of NDV.

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Tracheal tissues and blood samples were collected from twenty different districts of Punjab

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for virus isolation and for immune status evaluation, respectively (Table 1 and Fig 1B). One

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hundred and one flocks were tested for presence of vNDV by passaging tracheal samples into

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9-10 day old chicken eggs free from maternal antibodies against NDV. Allantoic fluids were

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tested using a spot haemagglutination (HAI) test and viruses from positives samples were

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isolated and sequenced as described (5). Viral RNA was extracted, cDNA was synthesized

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and sequenced as previously described (6) (10).

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influenza were used as previously described (7). For sequence analysis of vNDV primers for

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amplification

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CCCATTAGAGGCATACAACAG

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evolutionary relationship among these isolates and other GenBank sequences was determined

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using MEGA6 software (8). All the F gene sequences obtained in this study were deposited in

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GenBank under the Accession Number KF761533 to KF761552 (Table 1). For

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seroprevalence of NDV antibodies, a total of 320 random blood samples from commercial

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farms of each different district were collected, and the Geometric Mean Titres (GMT) and

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standard deviations were calculated (7).

of

a

374

bp

region and

of

the

Universal primers specific for Avian

Fusion

gene

were

designed

R- CAATATAGGGTAGCCGGTGAT).

(FThe

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Status of poultry flocks. All the farms studied practiced routine vaccination at the hatchery.

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In 80% of the farms, birds were vaccinated with a live LaSota vaccine at day 1 via spray or

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eye drop at the hatchery and at days 7 and 14 via water. Twenty per cent of the farms used an

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additional inactivated vaccine dose on day 24. Eighty eight out of 101 evaluated farms were

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found to be positive for the presence of NDV, irrespective of age (broiler and layers) based

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on spot HAI and the matrix PCR test, and of those 20 farms had confirmed presence of

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virulent viruses. The flocks were believed to be free from vNDV, however because NDV is

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endemic in Pakistan, the origin of the outbreaks are unknown. Mortality ranged from 6 to

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50% and morbidity rate was recorded to be up to 60% in some farms, despite vaccination. In

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most severe cases the disease erupted at 21-25 days of age and birds succumbed to death

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within 10-15 days (Table 1). No avian influenza was detected. Mortality due to vNDV

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occurred in both environmentally controlled houses and in open sheds (data not shown).

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Immune status assessed by measuring mean HI titers from serum samples (log2) showed high

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average values ranging from 4.7 to 6.9, and HI of log2 greater than 3 was detected in 99% of

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the 320 birds tested (Table 1).

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Molecular epidemiology. Twenty NDV isolates representative of the different districts were

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selected for genotyping (Table 1, Figures 1A, B and C). Partial and complete (F) gene

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sequences (374 and 1662 base pairs) of viruses from different districts (n=20) were obtained

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and compared with F gene sequences of representative NDV isolates belonging to genotypes

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of class II of NDV, and to previously characterized Pakistani viruses. Maximum likelihood

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phylogenetic analysis based on nucleotide sequences demonstrated that all 2013 NDV

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isolates grouped within genotype VII of class II as subgenotype VIIi (Figure 1A and B).

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Nucleotide sequence of this highly variable region of the F protein revealed 99.6 %

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nucleotide identity among 2013 isolates, and 96.4% identity to 2012 isolates of sub-genotype

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VIIi. All other available NDV GenBank sequences revealed lower nucleotide identity to the

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new isolates, suggesting that all of the 2013 viruses are related to each other and that they

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originated from viruses of the 2012 outbreak. We have recently shown that subgenotype VIIi

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has replaced the existing viruses of genotype XIII and VIId in Pakistan, and was the

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predominant subgenotype in poultry during 2011 and 2012 (9, 10). The virulence of the new

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isolates was confirmed by sequence of the predicted cleavage site of the F protein, which

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indicated that all the isolates carried the112-RRQKRF-117 motif corresponding to the cleavage

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site of virulent NDV.

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Significance. Sequence analysis confirms that one year after a highly virulent outbreak

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occurred in Pakistan, 20 vNDV positive farms in the vast geographic region of Punjab were

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infected with viruses of the same lineage that caused the problem in 2012. This infection

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occurred during a three months period (and continued during 2014) despite of the overall

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relatively high levels of anti NDV antibodies detected in 99% of the samples. Current

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knowledge on NDV vaccines based on experimental studies in laboratory settings that

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mimicked field conditions suggested that herd immunity of >85% with antibody titers of log2

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HI >3 should protect against viral spread (11). However the authors cautioned about the

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limitations of the study due to the reduced amount of data available and to the possible

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difficulties in achieving effective distribution of antibodies titers in large populations of

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poultry. Our results, although limited, present field data that suggest that despite high levels

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of anti NDV antibodies titers of > 3 log2 HI in 99% of the tested birds in different farms and

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localities there was very high incidence of the disease (Table 1). Although it is possible that

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correctly vaccinated chickens may not sustain vNDV under optimal laboratory conditions,

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other factors that may affect the immune status of the flock in the field, such as

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immunosuppression or non-uniform vaccination leading to a rolling infection, may allow the

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persistence of virulent viruses in vaccinated birds. Thus, there is need to re-evaluate the

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robustness of current vaccination methods under field conditions and the role of vaccinated

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birds in virus maintenance.

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Figure 1 legend: 1A. Phylogenetic analysis of the partial F gene distances between new and

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past virulent NDV Pakistani isolates based on the Maximum likelihood method. 1B.

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Phylogenetic analysis based on the full fusion gene of selected isolates. 1C. Pakistan map

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showing sites of disease incidence.

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Table 1: Virulent samples description, GenBank accession numbers, farm location, size, age

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of birds, observed mortality, mean and standard deviation of HI titers.

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Acknowledgements

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Funding has been provided by the US Department of State (USDA/ARS/BEP/CRDF) NDV

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31063, USDA CRIS 6612-32000-064 and PARB CGS project No.572. The team is thankful

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to the PARB for technical assistance during the surveillance studies, particularly the Director

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Poultry Research Institute, Rawalpindi, for assigning a team to facilitate the collection of

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samples.

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S t ra in Chic ke n/ P a k/ Ba h wa lpur / 300/ 2013 Chic ke n/ P a k/ Fa is a l a ba d/ 03/ 2013 Chic ke n/ P a k/ Gujr a nwa la / 589/ 2013 Chic ke n/ P a k/ Gujr a t / 234/ 2013 Chic ke n/ P a k/ Ha f i z a ba d/ 611/ 2013 Chic ke n/ P a k/ Ka s ur / 08/ 2013 Chic ke n/ P a k/ Kha ne wa l/ 55/ 2013 Chic ke n/ P a k/ La h or e / 02/ 2013 Chic ke n/ P a k/ Lod hr a n/ 62/ 2013 Chic ke n/ P a k/ Mult a n/ 43/ 2013 Chic ke n/ P a k/ Muz a f a r ga r h/ 160/ 2013

Ac c e s s io n No .

Ag e o f b i rd s La t i t u d e

Lo n g i t u d e

Fl o c k s i z e

( In D a y s ) / Ho s t

S a mple s c o lle c t e d

HI t i t r e P o sit iv e %

KF761551

29.3956

71.6836

36,000

22/ Br oile r

16

94

6

KF761538

31.418

73.079

30,000

28/ Br oile r

7

100

16

KF761536

32.15

74.1833

36,000

31/ La ye r

29

82

15

KF761548

32.5738

74.0789

35,000

35/ Br oile r

55

56

15

KF761535

32.07

73.68

6,000

21/ Br oile r

2

100

31

KF761540

31.117

74.4502

77,000

08/ Br oile r

17

53

40

KF761552

30.303

71.9333

15,000

12/ Br oile r

23

91

7

KF761537

31.545

74.3406

100,000

37/ Br oile r

16

100

50

KF761543

29.5333

71.6333

2,000

28/ La ye r

6

100

5

KF761541

30.1978

71.4697

8,000

42/ Br oile r

48

81

45

KF761549

30.0703

71.1933

22,000

21/ Br oile r

8

100

23

KF761546

31.45

73.7

9,000

25/ Br oile r

6

100

50

KF761550

32.2167

74.95

17,000

26/ Br oile r

17

81

33

KF761547

30.8091

73.4477

62,000

29/ Br oile r

9

100

27

Chic ke n/ P a k/ Na n ka na

S t . De v

Mo r t a l i t y % 5.7

1.35

6.0

0.97

6.9

1.06

6.0

0.85

5.4

1.30

5.7

2.12

6.0

0.89

5.8

2.44

5.7

0.98

6.8

1.65

5.4

0.81

6.8

1.69

S a hib/ 155/ 2013 Chic ke n/ P a k/ Nor wa l/ 40/ 2013 Chic ke n/ P a k/ Oka r a / 118/ 2013 Chic ke n/ P a k/ Ra w a lpindi/ 188/ 2013 Chic ke n/ P a k/ S a hi wa l/ 06/ 2013 Chic ke n/ P a k/ S a r godha / 212/ 2013 Chic ke n/ P a k/ S he i khpur a / 22/ 2013 Chic ke n/ P a k/ S ia l kut / 88/ 2013 Chic ke n/ P a k/ Ve h a r i/ 46/ 2013

KF761534

33.6

73.0333

16,000

22/ La ye r

4

67

40

KF761539

30.5833

73.3333

23,000

35/ Br oile r

7

100

43

KF761533

32.0836

72.6711

3,000

22/ Br oile r

3

100

20

KF761545

31.715

73.985

29,000

24/ Br oile r

14

57

10

KF761544

32.4972

74.5361

6,000

49/ Br oile r

7

100

7

KF761542

30.0419

72.3528

7,000

22/ Br oile r

6

100

7

5.3

1.13

6.5

0.63

4.8

1.13

6.4

1.75

5.1

0.81

5.0

0.73

5.6

1.09

5.9

0.85

Table 1: Selected samples location, description, accession number corresponding to sequenced region, farm location and size, age of birds, observed mortality, mean and standard deviation of HI titers.

Figure 1A

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KF761539/Chicken/Pak/Sahiwal/06/2013 KF761533/Chicken/Pak/Sargodha/212/2013 KF761538/Chicken/Pak/Rawalpindi/188/2013 KF761549Chicken/Pak/Muzafargarh/160/2013 KF761541/Chicken/Pak/Multan/43/2013 KF761543/Chicken/Pak/Lodhran/62/2013 98 KF761535/Chicken/Pak/Hafiz abad/611/2013 KF761536/Chicken/Pak/Gujranwala/589/2013 KF761538/Chicken/Pak/Faisal abad/03/2013 KF761551/Chicken/Pak/Bahwalpur/300/2013 KF761542/Chicken/Pak/Vehari/46/2013 KF761552/Chicken/Pak/Khanewal/55/2013 KF761546/Chicken/Pak/Nankana Sahib/155/2013 KF761548/Chicken/Pak/Gujrat/234/2013 KF761540/Chicken/Pak/Kasur/08/2013 85 KF761537/Chicken/Pak/Lahore/02/2013 VIIi KF761550/Chicken/Pak/Norwal/40/2013 98 62 KF761547/Chicken/Pak/Okara/118/2013 KF761545/Chicken/Pak/Sheikhpura/22/2013 KF761544/Chicken/Pak/Sialkut/88/2013 66 KF113352/Chicken/Pak/UVAS/212/2012 63 KF113353/Pheasant/Pak/Lahore/136/2012 KF113344/Chicken/Pak/Okara/103/2011 KF113342/Chicken/Pak/Lahore/50/2011 KF113351/Chicken/Pak/UVAS/211/2012 KF113350/Chicken/Pak/Lahore/200/2012 KF113339/Chicken/Pak/Lahore/30/2011 79 KF113340/Chicken/Pak/Lahore/32/2011 KF113341/Chicken/Pak/Lahore/43/2011 90 KF113343/Chicken/Pak/Gujranwala/56/2011 KF113345/Chicken/Pak/Gujranwala117/2011 KF113346/Chicken/Pak/Khyber Puk. Khawa/118/2011 KF113347/Chicken/Pak/Khyber Puk. Khawa/119/2011 KF113348/Chicken/Pak/Khyber Puk. Khawa/162/2012 KF113349/Chicken/Pak/Kasur/191/2012 AY288998/cockatoo/Indonesia/14698/1990 Ancestral JN986837/chicken/NL/152608/1993 Ancestral AY444497/moluccan/Indonesia/904/87 Ancestral 61 48 80 DQ858357/China/YG03/2006 VIIf 51 AY028995/China/CH/A7/96/2001 43 90 DQ485256/Chicken/China/Gu/2000 VIIe EF589134/China/H2/2007 87 95 DQ485269/China/Chi/Gu13/2004 VIId EF579731/Broiler/Shandong/YS/2006 87 DQ417112/China/SSX03/2003 VIIg 85 DQ485271/China/Gu14/2005 VIIb 63 DQ227247/China/SDD01/2001 67 99 259879777/Avian/Mauritania/2006 XVIII 30 515794290/Chicken/Ivory Coast/2007 JX393313/Culex/Indonesia/JKT/1979 Ancestral JN942041/Cockatoo/India/7847/1982 Ancestral 20 JQ267583/Chicken/IranEMM/3/2009 51 JQ267580/Chicken/IranEMM/6/2011 84 JQ267581/Chicken/Iran-EMM/5/2011 GU182323/Chicken/SPVC/Karachi/NDV/43/2008 97 KF113338/Chicken/Pak/University Diagnostic Lab/12/2010 JQ517285/Chicken/NDV/UDL8/2011 82 JN682190/Chicken/CP/Islamabad2/2010 66 GU182331/Chicken/SPVC/Karachi/NDV/33/2007 XIII GU182331/Chicken/SPVC/Karachi/NDV/33/2007 65 57 JN682191/Chicken/CP/Islamabad3/2010 95 JN682184/Chicken/BYP/Lahore/2010 JN682185/Chicken/CP/Rawalpindi1/2010 25 JN682186/Chicken/CP/Islamabad1/2010 74 JN682187/Chicken/BYP/Rawalpindi/2010 JN682210/Chicken/BYP//2010 70 JN682189/Chicken/CP/Rawalpindi/2/2010 49 78 JN682211/Chicken/CP/2010 JN682188/Chicken/CP/Attock/2010 64 JN627507/GD1003/373838901/2010 XII JN627508/GD450/373838903/2011 99 GU182329/Chicken/SPVC/Karachi/NDV/26/2005 JX186997/Chicken/Dominican Republic/867-2/2008 JX915242/Chicken/Dominican Republic/28138-4/1986 99 0.02

XVI

Figure 1B

100

99

85 KF113345.1-chicken-Pakistan-2011 KF113348.1-chicken-Pakistan-2012 KF113350.1-chicken-Pakistan-2012 KF113351.1-chicken-Pakistan-2012 KF113339.1-chicken-Pakistan-2011 JX532092.1-peacock-Pakistan-2012 JX854452.1-pheasant-Pakistan-2011 100 HQ697254.1-chicken-Indonesia-2010 KF792018.1-chicken-Israel-2011 KF792019.1-chicken-Israel-2012 KF792020.1-parrot-Israel-2012 93 KF113344.1-chicken-Pakistan-2011 KF113341.1-chicken-Pakistan-2011 KF113342.1-chicken-Pakistan-2011 HQ697257.1-chicken-Indonesia-2010 KF113347.1-chicken-Pakistan-2012 KF113349.1-chicken-Pakistan-2012 KF113343.1-chicken-Pakistan-2011 Chicken/Pak/Gujranwala/649/2013 Chicken/Pak/Gakkhar/609/2013 96KF792021.1-chicken-Israel-2013

KF767105.1-lory-Indonesia-1988 JN986837.1-chicken-Netherlands-1993 AY288998-cockatoo-Indonesia-1990 100

97 100

0.01

JX393313.1-mosquito pool-Indonesia-1979 KF767104.1-cockatoo-Indonesia-1988 KF767106.1-parrot-Indonesia-1976 AB605247.1-chicken-Bali-2007 100 JX193074.1-egret-China-2011 KF026013.1-chicken-Malaysia-2011 HQ697261.1-chicken-Bali-2010 100 HQ697255.1-chicken-Indonesia-2010 HQ697256.1-chicken-Indonesia-2009 88

Figure 1C