Molecular Characterization and Pathology of Virulent Newcastle Disease Virus Isolated from Broiler Chickens in District Lahore, Pakistan

Among the largest poultry producers in the world, Pakistan is now placed at the 11th position with its poultry sector growing at an impressive rate of approximately 7.5 per cent per annum over the last decade. Chicken meat is not only a cheaper source of quality nutrition; it has also the therapeutic potential due to higher antioxidant property of chicken with black meat (Sehrawat et al., 2021). Economics of poultry production can be improved by reducing input on health care (Jaiswal et al., 2014). Among the infectious diseases, RNA viruses remain potential threat for commercial poultry due to virus evolving nature. During 2006 and 2009, Newcastle disease (ND) was ranked as the 3rd most crucial poultry disease (Rehan et al., 2019). Since the development of an organized poultry sector in Pakistan, NDV has been devastating to the poultry industry for many years. Domestic poultry is highly susceptible to ND infection, responsible for severe outbreaks worldwide, resulting in million-dollar losses to farmers (Rahman et al., 2016). The Newcastle disease virus (NDV) or Avian orthoavulavirus 1 causes Newcastle disease, which is an economically important transboundary viral disease. Virulent strains of NDV are highly contagious and can be devastating, especially in immune-compromised chicken (Ganar et al., 2014).

Despite widespread vaccination and stringent biosecurity steps, ND outbreaks are also reported in vaccinated flocks (Etriwati et al., 2017) while its incidence is higher in non-vaccinated flocks. However, outbreaks in recent years have raised concerns regarding vaccination failure, which may occur due to incompatibility between field and vaccine strains or immunosuppressed birds which do not produce NDV protective titer (Mehmood et al., 2019). The broad host range of NDV in avian species has contributed to a substantial genetic variation of the virus and the emergence of NDV variants. In this respect, the isolation of novel NDV variants with different pathogenicity from diversified hosts indicates the virus evolution with subsequent disease transmission implications (Dimitrov et al., 2016). The phylogenomic studies found that 21 genotypes circulate in multiple avian species globally, with genotypes VII, XIII and XXI type of viruses were causing most outbreaks in developing countries including Pakistan (Rahman et al., 2020b). A reference index for AAvV pathotyping can be established through intracerebral pathogenicity index in one-day-old chickens, disease severity in susceptible hosts and analysis of fusion protein cleavage motifs (Rahman et al., 2019a). Very virulent NDV isolates have at least three basic amino acids i.e. arginine (R) or lysine (K) starting at positions 112-116 with phenylalanine (F) at position 117, while low virulent viruses have basic amino acids only at positions 113 and 116 with leucine (L) at position 117 (Alexander et al., (2012).

Due to frequent outbreaks of ND in commercial chicken flocks, poultry farmers have to bear heavy economic losses every year. Lahore is the second largest city of Pakistan and is located at 31.52°N 74.35°E. Established poultry disease diagnostic centers by the public and private sectors in Lahore were approached for convenient sampling. This study was designed to identify the current field circulating NDV based on F gene from diseased and sick chicken flocks. Furthermore, phylogenomic analysis was conducted to give an insight towards the genetic diversity of circulating indigenous strains and comparative analysis with the previously described strains. Pathology of field circulating NDV was recorded by monitoring gross and histopathological changes in the morbid tissues.

From September 2018 to August 2019, morbid tissue samples (trachea, lungs, intestine) of NDV vaccinated commercial chickens were aseptically collected and pooled from different broiler poultry farms (n=100) of the Lahore district (Pakistan). About 3-5 birds’ samples were collected from a single flock. After observing the pathological lesions, the collected samples were adequately labelled and divided into two parts; for reverse transcriptase-polymerase chain reaction (RT-PCR), samples were placed in an icebox and for histopathology, samples were preserved into 10% neutral buffered formalin. The whole laboratory work was done in molecular pathology laboratory and histopathology laboratory at Department of Pathology, University of Veterinary and Animal Sciences Lahore.

For molecular detection of NDV, tracheal tissue from each 3-5 birds of every flock was pooled and triturated in sterile phosphate buffer saline (PBS). Tissue homogenates were centrifuged at 5,000 g for 3 min and the supernatants were inoculated (0.2 ml) in a 9-day old chicken embryonated eggs with no maternal antibodies against ND. Harvested allantoic fluid was subjected to spot haemagglutination assay and then haemagglutination inhibition assay using known hyperimmune serum for NDV. The positive samples were subjected to RNA extraction, cDNA synthesis and RT-PCR as described by (Saad et al., 2021). Previously reported primer set (NDV-F 589 5'-GGAGGATGTTGGCAGCATT-3' and NDV-R 575S 5'- GTCAACATATACACCTCATC-3') were used to amplify NDV fusion gene fragment (589 bp) (Pang et al., 2002). RT-PCR amplification was conducted in a final volume of 25 µl, containing PCR master mix (2X, Thermo Scientific, United States) (12.5 µl), DNA template (2 µl), primer forward and reverse (1.5 µl, each) and PCR grade water (7.5 µl). A Veriti™ 96-Well Thermal Cycler (Applied Biosystems™, United States) was adjusted to 95°C for 2 min (pre-denaturation) followed by 35 cycles of denaturation (95°C for 10 sec), annealing (53°C for 30 sec) and extension (72°C for 45 sec) with a final incubation of 72°C for 5 min. The PCR product was analyzed by gel electrophoresis against a 100 bp DNA ladder (Thermo Scientific, United States).

The purified PCR products in four positive samples for NDV were subjected to the Sanger Sequencing method. The nucleotide sequences were placed in a public database (http://www.ncbi.nlm.nih.gov/GenBank accessed on July 2, 2021) to retrieve the genome sequences (n=66) of avian host Newcastle disease virus reported in different geographic regions worldwide. The obtained nucleotide and deduced amino acid sequences were aligned and analyzed through ClustalW on BioEdit version 7.2.5. The output file was subjected to a distance-based neighbour-joining model in MEGA-X software to construct the evolutionary tree. The reliability of the tree was based on 1,000 bootstrap replicates.

Gross pathological changes were recorded from morbid tissues collected from sick birds found positive for NDV by PCR. While formalin-fixed tissue samples were processed for histopathology and sections of 4 µm thickness were stained with routine hematoxylin and eosin (H&E stain) to get examined under a light microscope for the recording of microscopic changes (Śliżewska et al., 2019).

Molecular identification and genetic characterization of NDV
 
Poultry production remains under challenge by infectious diseases. Despite vigorous vaccination, Newcastle disease (ND) is one of the prevalent diseases in Pakistan causing economic losses to the farmers. NDV F gene is well recognized genetic marker and capable of generating evolutionary information. F gene in NDV requires activation by proteolytic cleavage for infectivity. OIE categories NDV as velogenic when multiple basic amino acid sequences are present at cleavage sites of position 112-116 and phenylalanine (F) at position 117 in the F gene amino acid chain (OIE, 2012). Based on RT-PCR results, 04/100 (4%) samples were found positive for NDV by amplifying the product size 589bp (Fig 1). Due to high genome sequence similarities, they were grouped into two isolates. The partial gene sequences of the present study were submitted to the NCBI-GenBank database under accession numbers MW295940 (Avian avulavirus 1 isolate chicken/Pakistan/PATH-IV/2019) and MW427214 (Avian avulavirus 1 isolate chicken/Pakistan/PATH-VI/2018). In this study, NDV isolates showed the presence of multibasic amino acid cleavage sequence (RRQKRF) in F gene (Fig 2).



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The phylogenetic analysis represented that all retrieved isolates were clustered into two classes i.e., class 1 (avirulent in chicken) and class ll (virulent in chicken). While NDV–class ll was categorized into 21 genotypes. The pairwise homology distance matrix analysis revealed that our isolates had a maximum percent identity matrix (nucleotide: 91.33% to 99.49% and amino acid: 96.43% to 100.00%) with NDV genotype VII (Table 1).



Current study isolates were grouped within genotype VII where most of the recent Pakistani NDV isolates have been reported (Fig 3). Percentage nucleotide similarity showed that one isolate has maximum similarity with Iranian isolate (MH481362) of genotype VII and sub-genotype l which is not earlier reported from Pakistan. While second NDV isolate of the current study showed maximum similarity with already NDV isolate reported from Pakistan in 2018 belonging to genotype Vll and sub-genotype i (Fig 4). In Pakistan, NDV genotype VII, XIII and XXI are primarily reported in a wide range of hosts. Apart from this, genotype VII is crucial because of its prevalence in the clinically healthy backyard and asymptomatic wild waterfowl (Rahman et al., 2019b). This highlights the possibility of introducing this strain through the transboundary trade of poultry flocks as well as a possible way of NDV spread through migratory birds.

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Gross and histopathological examination
 
The clinical sign observed in the birds representing NDV affected flock were greenish diarrhea, depression, anorexia, discharge from the oral cavity, respiratory discomfort, oculonasal discharge, torticollis and up to 40% mortality. Similar clinical signs were recorded in an experimental velogenic NDV infection to chicken by (Rahman et al., 2019c). Among the gross lesion observed, congestion and oedematous lungs were noted in NDV affected birds (Fig 5b). Trachea showing the scattered haemorrhages with exudate in lumen were most predominant macroscopic lesions in the respiratory tract (Fig 5c) which are consistent with earlier reports in chicken with velogenic viscerotropic NDV infection (Brar et al., 2017). Pinpoint haemorrhages were found on the mucosal tips of proventriculus papillae (Fig 5e). In the intestine wall, clearly demarcated button-like hemorrhagic ulcers were present (Fig 5a). The pathologic changes observed in the proventriculus and small intestine are the pathognomonic lesions of velogenic viscerotropic NDV strain (Fentie et al., 2014). Hemorrhagic caecal tonsils were present in the NDV positive birds (Fig 5d). The gross pathology in the present study suggested that the extent of lesions was more severe in the proventriculus, intestine, trachea, lungs and caecal tonsils which were also recorded in previous studies (Etriwati et al., 2017; Nakamura et al., 2014; Rahman et al., 2019a). In the histopathological examination of morbid tissues (Fig 6), tracheal tissue showed degeneration and necrosis of mucosal epithelium along with congested capillaries. In the lung parenchyma, there were infiltration of mononuclear cells were observed along with congestion in the lumen of parabronchi. Intestinal tissue showed sloughed villi epithelium and marked infiltration of lymphohistiocytes in the mucosa. (Brar et al., 2017) also reported the following lesions of velogenic NDV infected chickens; degenerative changes in tracheal epithelial cells, pulmonary congestion and sloughing of intestinal villi.

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ND live vaccines being currently used in Pakistan belongs to G-1I (Lasota) and G-III (Mukhteswar). However, there are reports of morbidity and mortality even in vaccinated poultry flocks (Etriwati et al., 2017; Mehmood et al., 2019; Perozo et al., 2012). (Rehmani et al., 2015) has found virulent NDV genotype VIIi in vaccinated chicken in Pakistan. In the present study, NDV of G-VIIi and G-VIIl in vaccinated flocks with a high degree of morbidity and mortality imparts significance to the development of ND vaccine from indigenous isolate for better immunity and regular monitoring of antibody titer so the virulent virus cannot hit the flock with low immunity against the diseases. It can be drawn from the study results that NDV killed vaccine belonging to genotype VII prepared from indigenous isolate should be included in vaccination regime for protection against the current circulating NDV belonging to G-VII.

It is the first to report NDV G VIIl isolated from broiler chicken in Pakistan to the best of our knowledge. The current study demonstrated the molecular characteristics and pathology of field circulating NDV. Based on the gross lesion, histopathological examination and amino acid sequence of F gene, it is elucidated that isolated NDV has the pathogenic potential of velogenic NDV strain. ND in vaccinated flocks with a high degree of morbidity and mortality imparts significance to develop ND vaccine from indigenous isolate for better immunity and strict biosecurity measures be adopted which otherwise can breach the bird’s immunity against the diseases. Robust research and development activities are still warranted to save huge losses in the poultry industry of Pakistan. Therefore, in disease-endemic countries, continuous surveillance of viral evolution should be ensured for disease control interventions.

This study was financially supported by the Higher Education Commission of Pakistan through PhD Indigenous Scholarships Scheme (PIN No.518-108421-2AV5-101).