Repeated isolation of virulent Newcastle disease viruses in poultry and captive non-poultry avian species in Pakistan from 2011 to 2016

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Virulent viruses of the panzootic Avian avulavirus 1 (AAvV-1) of sub-genotype VIIi were repeatedly isolated (2011–2016)from commercial chickens and from multiple non-poultry avian species in Pakistan. These findings provide evidence for the existence of epidemiological links between Newcastle disease outbreaks in commercial poultry and infections with virulent AAvV-1 strains in other avian species kept in proximity to poultry. Our results suggest that the endemicity of Newcastle disease in Pakistan involves multiple hosts and environments.

Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://

Keywords: Newcastle disease virus NDV APMV-1 Pakistan Captive birds Epidemiology Endemicity Avian avulavirus 1, AAvV-1.

1. Introduction

Newcastle disease (ND) is a highly contagious and fatal disease affecting poultry and a wide range of wild birds worldwide that is caused by infections with virulent strains of Newcastle disease virus (NDV) (Miller et al., 2010; Dimitrov et al., 2016c). Recently, the International Committee On Taxonomy of Viruses amended the taxonomy of genus Avulavirus of the Paramyxoviridae family. Newcastle disease virus and all avian paramyxoviruses 2-13 were renamed to avian avulavirus (AAvV) 1 to 13, respectively (https:// For the purpose of this paper and for consistency with previous publication, further below we have used the taxa name Newcastle disease virus. Despite intensive vaccination, endemicity of ND is a significant problem across Asia, Africa, and Central America. Recent reports have documented that some of the newly identified viruses of sub-genotype VIIi are rapidly spreading from Southeast Asia, into the Middle East, to Eastern Europe and North Africa and can cause mortality in poorly vaccinated poultry (Miller et al., 2015b; Rehmani et al., 2015; Dimitrov et al., 2016c). The presence of virulent viruses in vaccinated birds in commercial farms (Rehmani et al., 2015) and their constant evolution over time (Miller et al., 2009) suggest the existence of a high environmental viral load with continuous replication of these virulent NDV strains in endemic countries. However, the nature of the endemicity and the mechanisms of panzootic viral spread for NDV are largely unknown.

For the successful control of ND it is important to identify the factors that contribute to its endemicity. The spillover (defined here as the bi-directional transmission of closely related NDV) of NDV between poultry and wild bird species has been reported previously (Vidanovic et al., 2011; Cardenas Garcia et al., 2013; Ayala et al., 2016). Exotic birds kept in captivity and pet birds have also been infected with virulent NDV strains (Nolen, 2002; Pedersen et al., 2004) and are considered a biosecurity threat to domestic and commercial chickens. Avian influenza studies have identified wild bird species that could be considered “bridge hosts” for the transmission of viruses between poultry and wild birds (Caron et al., 2014; Caron et al., 2015). We have recently shown that backyard chickens are an important component in the circulation of genotype VII virulent NDVs in Bulgaria and Ukraine (Dimitrov et al., 2016b).

Pakistan presents a unique opportunity to study mechanisms of viral maintenance and spread in endemic countries. In Pakistan, ND was first detected in 1963 (Khan and Huq, 1963) and since then outbreaks have been observed repeatedly in both commercial and backyard poultry flocks. There is a wide variety of non-poultry bird species (both free-living and kept in captivity) in Pakistan; however, limited information is available concerning the potential role of these avian species in the dissemination of NDV. To understand the relationship among the circulating viruses and to identify the avian species and the husbandry systems that might contribute to ND endemicity, we have isolated and characterized NDV from different avian species and production systems in Pakistan over a five-year period. Here, we describe the repeated isolation of highly related virulent NDV strains from poultry, non-poultry species kept in captivity, and wild birds (chickens, pheasants, peafowls, pigeons, exotic parakeets [Australian parakeets locally known as Bajri], and Black Swan) at multiple locations and in different types and sizes of flocks in Pakistan between 2011 and 2016.


2. Materials and methods

2.1. Sample collection, clinical observation and pathogenicity tests

Samples from sick or dead birds from broiler and layer commercial poultry farms, along with additional information, were collected from flocks experiencing either above average mortality or appearance of ND-like clinical signs. The samples from nonpoultry species were collected from birds kept in captivity in zoo or farm exhibitions, as family pets, or as racing birds, except for one free-living pigeon. The samples from the Zoo Park were collected during routine visits by the Veterinary Officer from zoo birds with clinical signs of ND under quarantine at the zoo. Backyard samples were collected across different neighborhoods, no specific criteria for selection of study sites existed, and sampling was performed upon signals of increased poultry mortality by owners and field veterinarians. The samples background data (e.g. dates and locations of sample collection, host species, husbandry types, flock size and age of birds, number of dead birds) were collected using standard form (template is available upon request).

In total, between 2011 and 2016, 52 NDV (21 from non-poultry species and 31 from poultry) were isolated at the University of Veterinary and Animal Sciences, Lahore, Pakistan (UVAS). The viruses from poultry were isolated from either vaccinated commercial flocks or non-vaccinated backyard chickens. Detailed information on the isolates is presented in Supplemental Table S1. Twenty one of the isolates (marked with superscript “a” in Supplemental Table S1), that have not been already analyzed at UVAS, were submitted to the Southeast Poultry Research Laboratory (SEPRL) of the USDA in Athens, GA and 20 of them underwent evaluation to establish intracerebral pathogenicity index (ICPI) values following routine procedures (OIE, 2012). Following the same procedure, the pathogenicity of eight additional viruses was assessed by intracerebral inoculation of NDV- and avian influenza virus (AIV)-free 1-day-old chickens at UVAS (marked with superscript “b” in Supplemental Table S1).


2.2. RNA extraction and sequencing

For the samples analyzed at SEPRL viral RNA was isolated from the allantoic fluid using TRIzol LS reagent (Invitrogen, USA) and the QIAamp RNA viral mini kit (Qiagen, USA) and further processed by next-generation sequencing as reported by Dimitrov et al. (2017). Thirty one samples were analyzed at UVAS and viral RNA was extracted from infected allantoic fluid using the TRIzol LS reagent (Invitrogen, USA) following the manufacturer’s instructions. Sequencing of the complete coding region of the fusion (F) protein gene was performed as described previously (Munir et al., 2010; Miller et al., 2015a; Miller et al., 2015b).


2.3. Phylogenetic analyses

All available complete F-gene coding sequences of class II NDV (n = 1542) were downloaded from GenBank (Benson et al., 2015) as of September 2016 and analyzed together with the sequences obtained in the current study. A smaller dataset of closely related previously characterized NDV (n = 19) and the isolates sequenced here (n = 52), were further analyzed phylogenetically using MEGA6 (Tamura et al., 2013). For comparison purposes, five additional sequences of more distant genotype VII viruses were also added. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-3 model, selected by corrected Akaike Information Criterion, with 1000 bootstrap replicates as implemented in MEGA6 (Tamura, 1992). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 0.4724]). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 76 nucleotide sequences (Supplemental Table S2). Codon positions included were 1st, 2nd, 3rd, and non-coding. All positions containing gaps and missing data were eliminated. There were a total of 1662 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).


3. Results

3.1. Clinical signs and in vivo characterization

Number of dead birds and clinical signs varied widely in different flocks and species with some large vaccinated chicken flocks and non-vaccinated pet birds showing high numbers of survivors (Supplemental Table S1). Infected birds of different species were of different ages and isolated at different locations (Fig. 1). Infected birds presented with respiratory, neurological and/or enteric clinical signs typical for ND. In pheasants respiratory signs and greenish diarrhea were observed. Diseased peafowls showed nervous signs with torticollis, tremors, disorientation and weakness and a few birds also had wing and leg paralysis. Upon post mortem examination hyperemic and hemorrhagic spleens were found. In parakeets no clinical signs except sudden death were observed. Infected pigeons had tremor and torticollis that are typical for the disease in this species (Vindevogel and Duchatel, 1988). The infected Black Swan had depression and greenish watery diarrhea along with nervous signs including circular movements of the neck and head 4–5 h prior to death. Birds in vaccinated poultry flocks showed clinical signs typical for ND (Miller and Koch, 2013).

In pathogenicity studies performed with SPF chickens (Supplemental Table S1) 20 selected viruses presented ICPI assay values between 1.75 and 1.96, typical for velogenic NDV (Alexander and Swayne, 1998) and demonstrating that there was no increase in virulence of the NDV strains obtained from the non-poultry species. Host-related patterns relating to the ICPI values were not observed.

All of the viruses whose pathogenicity was tested in NDV- and AIV-free 1-day-old chickens caused severe clinical signs and death within 2–3 days post inoculation for all inoculated birds, also suggesting the presence of virulent NDV.

Fig. 1. Locations in Pakistan where the studied Newcastle disease viruses were isolated. The provided scale bar is not valid for the general map of Pakistan in the top left corner of the figure.


3.2. Molecular characterization and epidemiology

The amino acid sequence at the cleavage site of the fusion protein of all studied viruses was found to be typical for virulent NDV strains with three basic amino acids between position 113 and 116 and a phenylalanine at position 117 (113RQKR↓F117)(OIE, 2012) and consistent with the determined ICPI values. The distance and phylogenetic analyses compared the complete F-gene of these and other viruses that circulated previously in Pakistan. The nucleotide distance between most of the viruses from captive non-poultry species and those from poultry varied between 0.1% and 0.9% with some of the viruses being almost identical (0.1% to 0.3%). Based on specific criteria set by Diel et al.(2012) all of the studied isolates (bold fontin the phylogenetic tree) were classified as members of sub-genotype VIIi (Fig. 2). The constructed phylogenetic tree demonstrates the very close phylogenetic relationship between virulent NDV from poultry and viruses from pet or other non-avian species kept in captivity during a period of five years. Most of the NDV isolates from captive birds grouped in monophyletic branches together with the viruses from chickens. In addition, two clearly distinct branches (1.4% nucleotide distance) of viruses from different species were identified and highlighted in boxes in the phylogenetic tree (Fig. 2). While the first branch contained viruses isolated between 2011 and 2016 predominantly from pigeons and chickens, the second branch consisted of viruses mainly from peafowl and chickens from 2013 to 2015.


3.3. Accession numbers

The complete F-gene sequences (n = 52) of virulent NDV obtained in this study were submitted to GenBank and are available under the accession numbers KU862283 to KU862296, KX496962 to KX496967, KX791183, KY076030 to KY076044, and KR676389 to KR676404.


4. Discussion

Infection with virulent NDV in captive non-poultry species of birds has been previously reported in Pakistan and other countries; however, most studies have reported outbreaks that occurred over short period of time and were considered to be caused by accidental spillover from a poultry outbreak (Seal et al., 1998; Vijayarani et al., 2010; Shabbir et al., 2012; Cardenas Garcia et al., 2013; Kumar et al., 2013; Dimitrov et al., 2016c). Here, over a five-year period, we demonstrated the repeated isolation of genetically very closely related virulent NDV strains from domestic and commercial poultry and captive non-poultry birds. A limitation of this study is that it was not designed as classical epidemiological study. These viruses were mostly isolated from samples taken from locations with increased mortality of poultry or clinical signs of ND-like disease, and in a few instances were from farms and zoos with lower numbers of dead poultry (Supplemental Table S1). Interestingly, phylogenetic evaluation of the viruses isolated during this period demonstrates the existence of clades of highly related viruses infecting different species and different types of production systems (see highlighted boxes in phylogenetic tree, Fig. 2).

Fig. 2. Maximum likelihood phylogenetic tree of the fusion protein gene complete coding sequences of the Newcastle disease viruses isolated in Pakistan between 2011 and 2016 from poultry and non-poultry avian species. The tree with the highest log likelihood (−7775.3243) is shown. The Roman numerals presented in the taxa names in the phylogenetic tree represent the respective genotype for each isolate, followed by the GenBank accession number, host name (if available), year of isolation, strain designation and country of isolation. Embolden taxa were sequenced in the present study. Taxa enclosed in highlighted boxes have higher genetic distance between them (1.4% nucleotide distance between both groups in boxes). The species with larger font on the left side in each box represent the species with higher abundance in each group.

These new data point to a significant role of non-poultry species kept in captivity in the same geographic region as poultry on the circulation of NDV in Pakistan. It is unclear if each one of these cases corresponds to a specific spillover event from poultry farms or from other unknown reservoirs. However, the high similarity of sequences (above 99.7%) and the close distances between some poultry farms to sites of isolation in pet birds and backyard birds point to the existence of epidemiological connections (Fig. 1). The continuous circulation of NDV in non-poultry species suggests the need to develop additional control strategies that would include active surveillance in pet rearing sites and or sites in which exhibition birds and wild birds are kept in captivity (e.g. zoos and parks). Recently, wild birds species that are more likely to be in contact with poultry (“bridge hosts”) have been identified in avian influenza transmission studies (Caron et al., 2014). A similar type of study would be needed to better understand the dynamics of transmission of Newcastle disease viruses. The grouping of peafowl with poultry isolates during 2013–2015 suggests the interaction of these two groups of birds and is an example of an area where increased knowledge and biosecurity measures could be enacted to prevent the transmission of NDV between the two groups of birds. The demonstration of clinical signs and the first isolation of virulent NDV in a Black Swan suggest that the range of possible hosts may be extending.

The epidemiological situation observed in Pakistan is likely to be similar to that of many countries in the developing world. Asia, Africa and Latin America are currently undergoing extensive transformation on their protein production systems toward intensive poultry farming. Large farms without adequate biosecurity are often surrounded by existing rudimentary production systems such as backyard flocks or non-poultry avian species kept for other reasons. Among these, backyard poultry has played and still does play a significant role in the economy of the villagers in rural areas where it is primarily kept for the production of meat and eggs. The majority of backyard poultry farming in Pakistan consists of small-scale (10–15 birds) units. As they are predominantly free ranging, generally there is complete deficiency of biosecurity and good husbandry practices that could prevent spread of NDV. In addition, traditions such as keeping exotic pet birds as status symbol, cock fighting, and for hobbies (pigeon racing and shows) may facilitate the movement of infected pet birds. Although presently uncommon, similar strategies as those used in commercial poultry, such as vaccination, may contribute to the better control of ND. Vaccination is not likely to prevent viral replication; however, most existing vaccines are shown to reduce virus replication and shedding up to 2 logs in comparison to naïve birds, which would help decrease the amount of NDV shed into the environment (Dimitrov et al., 2016a).



We would like to acknowledge Tim Olivier and Dawn Williams-Coplin for their technical assistance. We appreciate critical and constructive reviews provided by three anonymous reviewers. This work was supported by the Department of State Biosecurity Engagement Program (BEP, NDV 31063), the Defense Threat Reduction Agency Cooperative Biological Engagement Program, USDA/ARS #685/FRCALL 12-6-2-0005, USDA CRIS 6040-32000- 064-00D.

064-00D. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.


Appendix A. Supplementary data Supplementary data

Supplementary data associated with this article can be found, in the online version, at 04.010.


This article was originally published in Preventive Veterinary Medicine 142 (2017) 1–6, This is an Open Access article under the CC BY-NC-ND license (

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