Isolation and genetic characterization of virulent strains of avian paramyxovirus-1 from multiple avian species in Azad Jammu and Kashmir 2017–2018

Abstract

Despite intensive vaccination, endemicity of Avian paramyxoviruses-1 (APMV-1) is a significant problem in developing countries in Africa, Middle East, and Asia. Given the importance of APMV-1 in poultry and multiple non-poultry avian species, it is important to continue surveillance programs, routine monitoring and characterization of field isolates in the region where viruses are endemic. The purpose of this study was to pathotyped and genetically characterized 21 APMV-1s isolated from multiple avian species reared in different regions of Azad Jammu and Kashmir (AJK). Phylogenetic analysis based on complete fusion (F) gene sequences showed that 17 APMV-1 isolates obtained from commercial poultry and backyard birds belonged to sub-genotype VIIi. Though, one pigeon-origin APMV-1 isolate was clustered in sub-genotype VIg and three in recently designated new sub-genotype VIm of genotype VI. The pigeon-origin isolates had the following two motifs ¹¹³-RKKR↓F-¹¹⁷ and ¹¹³-RQRR↓F-¹¹⁷, while all other isolates had the polybasic amino acid sequence ¹¹³-RQKR↓F-¹¹⁷ at the F-cleavage site, which is characteristic of virulent APMV-1 strains. These results are consistent with the five viruses that had intracerebral pathogenicity indices (ICPIs) of between 1.50 and 1.73, corresponding to a velogenic pathotype. The APMV-1s isolated from commercial poultry and backyard birds in this study showed low nucleotide distance (0.3–0.9%) and genetically closely related (> 97%) to viruses repeatedly isolated (2011–2017) from multiple avian species in other states of Pakistan. Strengthened surveillance programs in both commercial poultry and backyard flocks are needed to better assess the commercial–backyard bird interface and form a basis for evidence-based measures to limit and prevent APMV-1 transmission.

Introduction

Newcastle disease (ND) is highly contagious and fatal viral disease affecting multiple avian species worldwide [1]. The ICTV (International Committee on Taxonomy of Viruses) has generated three genera, Paraavulvirus, Metaavulavirus, and Orthavulavirus [2]. Viruses of genus orthoavulavirus and species Avian orthoavulavirus-1 (AOAV1) (formally designated as Newcastle disease virus (NDV) or Avian paramyxovirus-1 (APMV-1) used thereafter for the purpose of this paper) cause high morbidity and mortality in a wide range of both wild and domestic birds [2, 3]. Among the animal viruses, APMV-1 remains the largest contributor of economic losses to the world’s economy [4].

APMV-1 has enveloped and non-segmented single-stranded negative-sense ribonucleic acid genome with six encoding genes (3′-NP-P-M-F-HN-L-5′) that transcripts into six structural proteins [5]. On the bases of fusion gene (F-gene) sequences, the APMV-1s are classified into two major groups, class I and II [6]. Currently, APMV-1s of class II are categorized into eighteen genotypes (I–XVIII) containing both virulent and avirulent APMV-1 strains, while class I viruses contain a single genotype, the viruses of this genotype are usually avirulent to chickens and distributed in wild birds and poultry worldwide [7, 8]. APMV-1s of class II genotype are currently associated with ND outbreaks in many Asian countries, and a particular concern is causing high mortality in vaccinated birds [3, 9].

Endemicity of APMV-1s worldwide is a major challenge to global poultry production and at least four panzootics have been documented since it was first identified in the 1926 [10]. APMV-1 isolates of class II genotypes II to IV were responsible for the first panzootic during 1920s to 1960s [11]. Viruses of genotype V were associated with second panzootic in Europe during the late 1960s and 1970s [12]. While the third panzootic was caused by APMV-1s of genotype VIb in racing, show pigeons originated in the Middle East during the 1980s [13]. Viruses from genotype VII are associated with fourth ND panzootic, which began around 1985, having spread from Asia, Africa, Europe, and South America, and continues today [8, 12, 14]. Since 2011–2012, reports have documented that some of newly emerged viruses of genotype VII (VIIh and VIIi) were responsible for ND outbreaks in Pakistan, Indonesia, and Israel and are rapidly spreading to other Middle East, Asian, North Africa, and Eastern European countries [1, 15–19]. The virulent strains of sub-genotype VIIi have been obtained from multiple avian species in different epidemiological studies during 2011–2016 [1, 20, 21]. Pigeons are mainly susceptible to APMV-1 of genotype VI, also known as pigeon paramyxovirus-1 (PPMV-1). Since the emergence in the late 1970s, originated in Middle East, the PPMV-1 strains have evolved rapidly and currently divided into 11 sub-genotypes phylogenetically through F-gene [22–24]. PPMV-1 infection was reported in other avian species such as Columbidae like pigeon and doves. PPMV-1 has been spreaded worldwide and considered endemic in many countries [22, 25–29].

To our knowledge, this is a preliminary report of isolation of virulent APMV-1 strains from the Pakistan’s administerial self-governing state Azad Jammu and Kashmir (AJK). Despite the vigorous vaccination, ND outbreaks have been reported from all the areas of Pakistan since 1963 [30], affecting not only the commercial poultry but also wild and domestic birds. Historically, backyard birds including chicken, ducks, and geese have been suggested as the probable culprit in the maintenance and evolution of APMV-1 as generally these birds are not vaccinated in their expenses. Due to the lack of biosecurity measures, the backyard poultry suggested the potential risk as a source of infection for the commercial poultry, since these birds rearing freely around houses and as they may have much contact with the migratory birds [31, 32]. In past outbreaks (California 2002–2003 outbreak), backyard poultry and pet animals have been found to be infected with virulent APMV-1 strains, and therefore, it is believed that these birds present a considerable risk to poultry industries [33, 34]. It is, therefore, important to identify components of the agro-ecosystems that may contribute to the maintenance and dissemination of the disease.

To better understand the current epidemiology of the disease in AJK, we sampled multiple avian species from various geographical regions during 2017–2018. We investigated the evolution and phylogenetic characterization of APMV-1s by employing the complete coding sequences of the fusion gene.

Material and method

Samples collection and virus isolation

Between January 2017 and March 2018, pooled oropharyngeal and cloacal swabs (n = 239) were collected from multiple avian species including chickens (Gallus Gallus domesticus), geese (Anas anser domesticus), ducks (Anas platyrhnchos domesticus), peacocks (Pavo cristatus), and pigeon’s (Columba livia domesticus) flocks in AJK. All samples were collected from three different locations: Mirpur, Kotli, and Bhimber districts of AJK (Fig. 1). The collected pooled samples were immediately transferred to 1 ml transport medium PBS, pH 7.0 contained 200 mg/ml streptomycin, 2000 U penicillin, 250 mg/ml gentamycin, 2.5 mg/ml amphotericin B in the field. The samples were kept cool and transported into the Molecular Biology and Biotechnology Laboratories, Virtual University of Pakistan, Lahore using thermal box with ice. The viruses were inoculated into 9–11-day-old embryonated chicken eggs (ECE) (free from APMV-1 specific antibody). The infectious allantoic fluids were harvested at 48 or 96 h post inoculation and analyzed by hemagglutination assay (HA) and hemagglutination inhibition assay (HIA) using the previously described method [35].

Fig. 1.

Map of Azad and Jammu Kashmir (AJK) showing the geographical regions where samples were collected

Pathogenicity study

Assessment of the pathogenicity of five selected APMV-1 isolates was done by the intracerebral pathogenicity index (ICPI) assay using HA-positive allantoic fluids in one-day-old Hubbard chickens (free from specific APMV-1 antibody) according to the previously established protocol [35].

RNA extraction, F-gene amplification, and sequencing

Genomic viral RNA was extracted from 250 μl HA positive allantoic fluids by TriZol LS-reagents (Invitrogen, USA) according to manufacturer’s procedure. Next, 10 μl of eluted RNA was used for complementary DNA (cDNA) using transcriptor first strand cDNA synthesis kit® (Thermo Scientific, USA) according to manufacturer’s protocol. Extracted RNA and synthesized cDNA were quantified using the Nanodrop spectrophotometer (Thermo Scientific) and stored at − 86 °C until further use. The complete F-gene (1662 base pair [bp]) was amplified and sequenced through previously reported primers [1]. F-gene amplification was performed using Superscript-III One-Step RT-PCR system with Platinum Taq DNA polymerase enzyme (Invitrogen, Carlsbad, CA) as per recommendations. The amplificons were run on 1.2% agarose gel electrophoresis and purified using Quick Clean DNA gel extraction kit (Qiagen, Val, CA). The purified F-gene products were sequenced by ABI 3130 automated sequencer (ABI, Inc., Foster City, CA).

Phylogenetic analysis

BioEdit software 7.2.5 was used for nucleotide sequences editing, assembly, and analysis [36]. The coding region of F-gene was used to construct phylogenetic trees in order to infer the evolutionary history of APMV-1 isolates with other representatives of class II genotypes (total n = 94 F-gene sequences for genotype VII and n = 92 for genotype VI). The F-gene sequences of various genotypes included in the phylogenetic analysis were retrieved from the GenBank. The evolutionary history of the isolates was inferred using the maximum likelihood method based on the general time-reversible model using MEGA software v7 [37]. Specific amino acid motif sequences associated to virulence of APMV-1 were evaluated in the coding region of F-gene.

Nucleotide sequences accession number

Complete F-gene of the currently APMV-1 isolates was submitted into GenBank with accession number MH717055 to MH717073, MH891147, and MH891148.

Result

Virus isolation and biological characterization

Twenty-one APMV-1 strains isolated from commercial and backyard chickens, peacocks, ducks, geese, and pigeons in different regions of AJK were studied here (Table 1). Out of 105 oropharyngeal and cloacal swabs from healthy and diseased commercial poultry flocks inoculated in ECE, six contained live APMV-1 viruses. The infected birds showed typical APMV-1-like clinical signs varied widely in various flocks, in most birds with respiratory and greenish diarrhea were observed. A total of 41 oropharyngeal and cloacal swabs were collected from healthy backyard chickens and propagated in ECE, and five of them were confirmed as APMV-1 by HIA and RT-PCR. Out of thirty-one ducks, and twenty-three geese were sampled rearing with or near backyard poultry in various locations, three clinically healthy ducks, and two geese were found to be positive for APMV-1 by virus isolation. APMV-1 was detected in a single peacock, out of eleven collected oropharyngeal and cloacal swabs. ND positive peacock displayed nervous signs such as tremors, depression, and torticollis accompanied with partially wings and legs paralysis. Out of twenty-eight oropharyngeal and cloacal swabs from clinically healthy and infected pigeons, four clinical samples were found to be positive for PPMV-1. The infected pigeons presented with neurological signs, torticollis, and tremors that are typical for the ND disease in this species. One diseased pigeon was observed with wing and leg paralysis and greenish diarrhea. ICPI assay was conducted on selected five APMV-1 isolates, and the value > 1.5 was observed classified as velogenic APMV-1 strains. Two APMV-1 strains isolated from commercial poultry, chicken/AJK/AW-c25 (1.61), chicken/AJK/AW-c26 (1.71), one from duck, duck/AJK/AW-d43 (1.50), one from peacock, peacock/AJK/AW-pc-45 (1.73), and one from pigeon, pigeon/AJK/AW-p53 (1.50) (Table 1).

Table 1.

Description of samples collected in different regions of Azad and Jammu Kashmir (AJK) during 2017–2018

Isolates	Accession number	Location	Date	Host	Flock size	Age	Mortality (%)	Geno	ICPI	Cleavage site
Chicken/AJK/AW-c21/2017	MH717055	Bhimber	Nov.2017	Broiler	1000	20D	10	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-c22/2017	MH717056	Bhimber	Nov 2017	Broiler	500	15D	00	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-c23/2017	MH717057	Kotli	Dec.2017	Broiler	50	12D	10	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-c24/2017	MH717058	Bhimber	Jan.2018	Broiler	100	10D	20	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-c25/2018	MH717059	Mirpur	Feb 2018	Broiler	20	36D	05	VIIi	1.61	RRQKR↓F
Chicken/AJK/AW-c26/2018	MH717060	Bhimber	March.2018	Broiler	50	37D	00	VIIi	1.71	RRQKR↓F
Chicken/AJK/AW-by31/2017	MH717061	Kotli	March.2018	Backyard	50	06Y	30	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-by32/2017	MH717062	Bhimber	Dec 2017	Backyard	25	03Y	10	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-by33/2017	MH717063	Bhimber	Feb 2018	Backyard	15	04Y	20	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-by34/2018	MH717064	Bhimber	Feb 2018	Backyard	15	05Y	50	VIIi	NA	RRQKR↓F
Chicken/AJK/AW-by35/2018	MH717065	Mirpur	March.2018	Backyard	20	02Y	50	VIIi	NA	RRQKR↓F
Duck/AJK/AW-d41/2018	MH717066	Kotli	Nov 2017	Duck	10	04Y	0	VIIi	NA	RRQKR↓F
Duck/AJK/AW-d42/2018	MH717067	Bhimber	Dec 2017	Duck	5	04Y	0	VIIi	NA	RRQKR↓F
Duck/AJK/AW-d43/2018	MH717068	Bhimber	Dec 2017	Duck	8	02Y	0	VIIi	1.50	RRQKR↓F
Geese/AJK/AW-g91/2018	MH891147	Bhimber	Jan 2018	Goose	31	05Y	0	VIIi	NA	RRQKR↓F
Geese/AJK/AW-g92/2018	MH891148	Bhimber	Jan 2018	Goose	11	03Y	0	VIIi	NA	RRQKR↓F
Peacock/AJK/AW-pc45/2018	MH717069	Kotli	Feb.2017	Peacock	5	04Y	30	VIIi	1.73	RRQKR↓F
Pigeon/AJK/AW-p51/2018	MH717070	Bhimber	March.2018	Pigeon	300	01Y	20	VIm	NA	RRKKR↓F
Pigeon/AJK/AW-p52/2018	MH717071	Kotli	March.2018	Pigeon	50	1.6Y	10	VIm	NA	RRKKR↓F
Pigeon/AJK/AW-p53/2018	MH717072	Mirpur	March.2018	Pigeon	20	02Y	10	VIm	1.50	RRKKR↓F
Pigeon/AJK/AW-p54/2018	MH717073	Bhimber	March.2018	Pigeon	15	1.4Y	5	VIg	NA	RRQRR↓F

Molecular diversity of APMV-1s

Seventeen APMV-1 isolates from various regions of AJK obtained in this study from commercial and backyard chickens, domestic ducks, geese, and the peacock were clustered in virulent sub-genotype VIIi, genotype VII, class II. The phylogenetic analysis was performed to compare the complete F-gene sequences (1662 bp) of the currently isolated APMV-1s and to the one from previously characterized Pakistani viruses isolated during 2011–2017 (n = 94, Fig. 2).

Fig. 2.

Phylogenetic tree constructed based on the complete fusion gene sequences of the APMV-1s isolated in AJK obtained from chickens, ducks, geese, and peacock (the isolates sequenced in this study are designated with) and the isolates representing other genotypes of class II. The evolutionary history was inferred by using the Neighbor-Joining algorithm based on general time reversible model with 1000 bootstrap replicates as implemented in MEGAv7.0. Five sequences representing each genotype were used and the analyses involved 94 nucleotide sequences of complete fusion gene. There were a total of 1661 positions in the final dataset

The nucleotide distances between the studied sequences and with previously characterized Pakistani strains available in GenBank were estimated. Most of the studied sequences were genetically very closely related to each other (0.1 and 0.4%). These viruses were found most closely related (0.3 to 1.4%) to sequences from viruses isolated from a variety of species (chickens, parakeets, ducks, geese, pheasants, peacocks, and pigeons) from Pakistan in 2011 and 2017. The phylogenetic analyses demonstrated the close phylogenetic relationship between the virulent APMV-1 isolated from the commercial and backyard poultry and viruses from non-poultry avian species during a period of 8 years. To further confirm the evolutionary relationship between the APMV-1 sequences studied here and sequences available in GenBank, phylogenetic analysis using the complete fusion gene sequences (1662 bp) was performed. In the phylogenetic tree, the isolates studied here grouped together within sub-genotype VIIi with the viruses that previously characterized in other provinces of Pakistan (Fig. 2).

All the isolates were predicted to contain the motif of ¹¹³RQKR↓F¹¹⁷ at the cleavage site of fusion protein classified as velogenic in nature. In order to determine the phylogenetic relationship between the four PPMV-1 sequences obtained from clinically healthy and diseased pigeons with other NDV isolated worldwide, the complete F-gene sequences (1662 bp) obtained in the present work along with other related sequences used to construct a phylogenetic tree (n = 92). The phylogenetic analysis demonstrated that all the four sequences obtained from pigeons studied here were clustered with viruses of genotype VI. Based on the analysis of complete fusion gene sequences, the genotype VI currently divided into at least twelve sub-genotype, namely VIa–VIm [22, 24]. In the current work, one PPMV-1 sequence obtained was clustered with previously described viruses into sub-genotype VIg, and three isolates were grouped into a distinct sub-genotype VIm (Fig. 3).

Fig. 3.

Phylogenetic tree constructed on the basis of complete fusion gene sequences of APMV-1 isolates obtained from pigeons and other APMV-1s isolates representing other genotype and sub-genotypes of class II. The four strains in this study are designated with. The evolutionary history was inferred by using the Neighbor-Joining algorithm based on general time reversible model with 1000 bootstrap replicates as implemented in MEGAv7.0. Three sequences representing each genotype were used and the analyses involved 92 nucleotide sequences of complete fusion gene. There were a total of 1659 positions in the final dataset

The pigeons infected with isolates pigeon/AJK/AW-p54/2018 (VIg) and pigeon/AJK/AW-p53/2018 (VIm) showed depression, greenish diarrhea, and neurological signs, and mortality was observed in the flock before sampling. The other two isolates pigeon/AJK/AW-p51/2018 and pigeon/AJK/AW-p52/2018 were obtained from clinically healthy pigeons with no obvious clinical manifestation. The evolutionary distance between sub-genotype VIg obtained in the current work with the rest of recently described Pakistani viruses was 0.2 to 0.5%. However, these viruses together with other previously described Pakistani viruses clustered together in a separate monophyletic branch with sub-genotype VIg and showed higher genetic diversity (4.7–4.8%) to the rest of sub-genotype VIg viruses. The currently isolated pigeon-derived sequences of sub-genotype VIg are genetically closely related to viruses from pigeons in Punjab province of Pakistan, Nigeria, Egypt, Ukraine, Russia, and Kazakhstan isolated during 2005 to 2016 [22, 29, 38, 39]. Three isolates (AW-p51, AW-p52, and AW-p53) obtained in this work was grouped together with other Pakistani isolates within newly designated sub-genotype VIm [22]. The genetic distance of these viruses to the rest of viruses in sub-genotype VIm was 0.1–0.3% which shows much closed phylogenetic relationship. The fusion protein cleavage site of three isolates (pigeon/AJK/AW-p51/2018, pigeon/AJK/AW-p52/2018, and pigeon/AJK/AW-p53/2018) was ¹¹³RKKR↓F¹¹⁷ and one isolate (pigeon/AJK/AW-p54/2018) with different motif ¹¹³RQRR↓F¹¹⁷, characteristic of typical virulent APMV-1 strains.

Discussion

This is the preliminary report of isolation and molecular characterization of field APMV-1 isolates from commercial and backyard chickens (Gallus Gallus domesticus), ducks (Anas platyrhynchos domesticus), geese (Anser anser domesticus), peacocks (Pavo cristatus), and pigeons (Columba livia domestica) in Azad Jammu and Kashmir (AJK) during 2017–2018. ND has always noticed a great and prominent problem in the poultry industry of AJK with other respiratory infections. Despite the extensive use of ND vaccination, the outbreaks occur sporadically throughout the year in the commercial poultry flocks as well as other non-poultry avian species are greatly affected by APMV-1. Because of the ever-growing population and extensive need of transformation on their protein production system, AJK leads toward high commercial poultry farming. The commercial poultry farms are always surrounded by backyard birds with other non-poultry avian species kept for other reasons. Backyard birds including native chickens, ducks, geese, turkeys, and pigeons are raised mostly by low-income households engaged in small flocks of 5–20 birds based on traditional methods. The backyard birds are mainly kept for eggs and meat as their main source of animal protein. The peacocks (Pavo cristatus) belong to the family Phasianidae in the order Galliformes. They are primarily native to many Asian countries and generally raised together with other backyard species in houses as a pet animal. The present study was conducted to isolate and phylogenetically characterized APMV-1s obtained from different geographical locations of AJK to evaluate the epidemiological relation to other circulating APMV-1s in other regions of Pakistan. The obtained APMV-1 isolates were biologically and genetically characterized on the bases of ICPI and complete fusion gene sequences. Based on the presence of the polybasic amino acid sequence ¹¹³RQKR↓F¹¹⁷ at the fusion protein cleavage site, all the APMV-1 strains isolated from commercial and backyard chickens, geese, ducks, and peacock were classified as velogenic in nature. The cleavage motif of the APMV-1 isolates obtained from pigeons had the following two motifs, ¹¹³RKKR↓F¹¹⁷ and ¹¹³RQRR↓F¹¹⁷. All of these motifs are characteristics of virulent APMV-1 [35]. The ICPI test was performed for selected viruses and determined values above 1.5 that are indicative for virulent APMV-1 (Table 1) [35].

In this study, we demonstrated the isolation of genetically very closely related virulent strains of APMV-1 of sub-genotype VIIi from multiple avian species including commercial and domestic poultry, ducks, geese, and peacock in various regions of AJK. Furthermore, these viruses were phylogenetically very closely related to previously characterized virulent strains of Pakistani APMV-1 isolated from various poultry and non-poultry avian species during 2011–2016 [1, 20, 21]. A possible study limitation is that it was not designed as an appropriate epidemiological study, and the studied viruses were mostly collected from locations with mortality reported or samples taken from bird/flocks with clinical signs of ND like disease. A phylogenetic analysis revealed the isolation of highly related viruses infecting various poultry and non-poultry avian species (Fig. 2).

These data point to a significant role of domestic poultry and various non-poultry avian species kept near to commercial poultry production facilities in the maintenance and circulation of these viruses. In the present study, high similarity of sequences (> 98%) obtained from multiple avian species pointed to the existence of epidemiological connections. Wajid and co-authors have recently reported the simultaneous circulation of highly related sub-genotype VIIi virulent APMV-1 in various poultry and non-poultry avian species in Pakistan over a 5 years period (2011–2016) [1].The investigation of the epidemiologic link between highly related APMV-1s is highly significant particularly in closely located geographical regions where ND has acquired endemicity. Backyard poultry may serve as an amplification host as they are of great concern to be exposed to virulent strains of APMV-1 because of the low biosecurity and lack of vaccination [40]. They increase the probability of virulent APMV-1 transmission to commercial poultry production facilities, because of widespread presence in close proximity of poultry farms, high chance of contact to wild birds, and/or a combination of these and other environmental factors. Surveillance efforts in these backyard birds may be expedient to gather data on circulating viruses and their pathogenicity. The isolation of virulent APMV-1 strains circulating in backyard chickens in Ukraine and Bulgharia during 2002 to 2013 suggests the possibility of a domestic or urban cycle of maintenance [41]. APMV-1 in backyard flocks that spilled over to commercial poultry production facilities was linked to the most recent ND outbreak in the USA [40]. Waterfowl are the persistent host and considered the natural reservoir of harboring both virulent and avirulent APMV-1s [10]. In the present study, two virulent APMV-1 strains (duck/AJK/AW-d41/2018 and duck/AJK/AW-d42/2018) obtained from clinically healthy ducks (Anas platyrhynchos domesticus) without showing clinical signs or apparent symptoms. The duck/AJK/AW-d41/2018 strain was isolated from clinically healthy duck rearing with backyard chickens in a house. Two other virulent strains of APMV-1 (AW-g91/2018 and AW-g92/2018) isolated from clinically asymptomatic geese kept in close proximity to other backyard birds are found positive with genetically closely related APMV-1 viruses studied here. AJK is highly populated and a small state with significant numbers of pet birds are in close proximity to poultry farms raising the possibility that short distances may have played a role in the spread of the ND. The virulent strains of APMV-1 have been isolated from diseased peacocks from national zoos in Pakistan during 2012–2015, displayed nervous signs with torticollis, disorientation, and weakness prior to death [1]. In this work, peacock/AJK/AW-pc45/2018 isolate was obtained from diseased peacock in Kotli region, AJK with severe neurological signs such as depression, tremors, and torticollis accompanied with partially wings and legs paralysis consistent with velogenic APMV-1.

Pigeons are not migratory, they are synanthropic birds or they may breed for meat and also as a pet companion birds. Pigeons may be infected with all genotypes of APMV-1, but genotype VI is considered as the main cause of Newcastle disease in family Columbidae including various species of domestic and wild pigeons as well as doves [42]. The similar viruses are isolated from non-Columbidae such as domestic fowl (Phasianidae, Gallus species), waterfowl (Anseriformes), partridges (Phasianidae), and robins (Turdidae) which has been considered a global threat [43, 44]. Wajid et al. [2017] had isolated sub-genotype VIIi (commonly found in non-Columbidae) from healthy pigeons that demonstrated the additional culprit and indemnity of maintenance and transmission of virulent APMV-1 strains [1]. However, the transmission of pigeon-derived APMV-1 viruses to poultry was reported on a few events in European countries during the past decades [11]; however, the potential of these viruses to cause clinical disease in poultry must not be underestimated. In the current work, the four PPMV-1s obtained from their repositories in AJK were fully characterized after propagation and confirmed by HI and RT-PCR. The phylogenetic analyses demonstrated that one PPMV-1s (AW-p54/2018) studied here clustered with viruses of sub-genotype VIg with previously described viruses isolated from pigeons in sex distant countries including Egypt, Nigeria, Russia, Ukraine, Kazakhstan, and Pakistan during 2005 to 2016 (Fig. 3) [22]. In addition, three PPMV-1 viruses studied here were clustered into newly designated sub-genotype VIm recently detected in Pakistan between 2014 and 2016. While both groups of viruses are simultaneously circulating, viruses from the new sub-genotype VIm are now found more frequently causing ND outbreaks in pigeons. Though, the mechanism of spread of genetically closely related viruses at long distances remains unknown. A potential probability might be caused by migratory birds on a single or multiple occasions or trade of live pigeons among these unrestricted distant regions. The cases in pigeons have been identified virtually throughout the year since 2014 in other regions of Pakistan [22, 23], the dissemination to AJK state was not unexpected.

However, a significant problem in AJK is the great density of farms with different bird species living in a close proximity suggesting the short distances among the domestic birds to commercial poultry farms may have played a role in the maintenance and spread of virulent APMV-1s. Numerous earlier studies demonstrated the spillover of virulent and avirulent APMV-1s between wild and domestic birds and commercial poultry [1, 6, 44, 45]. The situation in the region is very much similar to other Asian, Latin America, and African countries that are currently establishing intensive poultry farming. Most countries have common characteristics of mixed poultry production systems that may favor the maintenance and spread of viruses [1, 46]. On the basis of previous experience, the movement of live birds without quarantining across/within states of country and intermingling of multiple avian species in live poultry markets from various sources are likely to increase the virus transmission and evolution [47].

To our knowledge, this is the first description of virulent APMV-1 isolates obtained from clinically healthy and diseased commercial and backyard chickens, peacock, domestic ducks, geese, and pigeons in AJK. This study provides important information about the epidemiology and genetic relationship of APMV-1 viruses in poultry and non-poultry avian species. We are suggesting the trading birds for different purpose and bird shows certainly allows the spread of new APMV-1 strains to susceptible birds, except strict control measures are applied. The APMV-1 viruses in present work are genetically closely related to viruses from other geographical regions of Pakistan, and their presence suggests the potential risk of APMV-1 being brought into the region, possibly leading to bird infections. In AJK, isolation of virulent strains of APMV-1 from multiple avian species and present results highlight prevalent and consistent evolution in circulating APMV-1s which demonstrated consistent surveillance studies of the viruses in other regions.

Funding information

This work was supported by Office of Research Innovation and Commercialization, Virtual University of Pakistan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This research work on animals was approved by the Departmental Ethical Research Committee of the Virtual University of Pakistan, with approval number 005–16.