Molecular Characterization of Class II Newcastle Disease Virus from Field Outbreaks in Mizoram, India

Newcastle disease is one of the highly contagious infectious diseases of poultry because of its worldwide distribution and the potential for devastating economic losses. It occurs in at least six of the seven continents of the world and is enzootic in many countries including India. The disease is caused by Avian Paramyxovirus I (APMV-1) or Newcastle disease viruses (NDV) under the genus orthoavulavirus subfamily Avulavirinae of the family Paramyxoviridae (ICTV, 2019). The virus is a negative sense, non-segmented and enveloped with a single-stranded RNA genome of approximately 15.2 kb (Cao et al., 2013; Zhang et al., 2012). The NDV genome is composed of six genes that encode their corresponding six structural proteins: Nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN) and the RNA polymerase (L) (Chambers et al., 1986). RNA editing of the P protein produces additional non-structural proteins V and possibly W (Chambers and Samson, 1982; Collins et al., 1982; Locke et al., 2000; Mebatsion et al., 2001; Steward et al., 1993). The F glycoprotein which is responsible for the viral particles to fuse with the host cellular membrane is postulated as the main determinant of the viral pathogenicity (Gotoh et al., 1992; Ogasawara et al., 1992). The World Organisation for Animal Health (OIE) has defined the virulent strains of NDV that have an intracerebral pathogenicity index of 0.7 or higher (2.0 is maximum) or a fusion cleavage site starting from amino acid position 112 with multiple basic amino acids and phenylalanine at position 117 (OIE, 2012).
 
Based on the consensus amino acid sequences at F gene, NDV is divided into class I NDV and class II NDV strains. The class I NDV strains are grouped into a single genotype and 3 sub-genotypes and class II NDV strains, divided into at least 21 distinct genotypes (I-XXI) made up of several sub-genotypes (Dimitrov et al., 2019). NDV viruses of class I are mainly composed of avirulent strains, usually circulating among wild birds, whereas viruses of class II are more frequently virulent (Miller et al., 2010) and mainly isolated from poultry.
 
Class II NDV are also categorised into five pathotypes based on clinical signs in infected chickens and are designated: a) viscerotropic velogenic (VVND), b) neurotropic velogenic (NVND), c) mesogenic, d) lentogenic or respiratory and e) aymptomatic. Keeping in view the divergence of the circulating viral strains and their transboundary emergence, molecular characterization studies are essential in order to keep track of the evolution and genetic diversity of the viral strains. In the present study, we have recorded severe field outbreaks of NDV, isolated the circulating strain and characterized them based on complete F gene analysis.

Study areas and sample collection
 
The outbreak of ND was investigated in both backyard and commercial poultry farms located in Aizawl districts of Mizoram during the period of 2021 to 2022. A total of 25 poultry farms comprising a total population of 2165 birds with complaints of mortality associated with respiratory disease, severe depression and greenish-to-whitish diarrhoea were studied. The affected birds were clinically examined at the farm premises. A detailed post-mortem examination was conducted on a total of 121 dead birds from affected farms and the gross lesions were recorded. Representative tissue samples comprising of trachea, lungs, proventriculus, spleen and caecal tonsils were collected in 10% buffered formalin for histopathological examination and also preserved at -80°c for molecular diagnosis. Formalin-fixed tissues were subjected to histopathological processing and staining following standard method (Bancroft and Gamble, 2008). Hematoxylin and Eosin-stained individual sections were microscopically examined and the histopathological changes were recorded. The entire work was carried out in the Department of Veterinary Pathology, C. V. Sc. and A. H., CAU(I), Selesih, Aizawl, Mizoram.
 
Isolation of newcastle disease virus
 
The RT-PCR positive tissue samples were used to prepare the inoculums for isolation of the NDV field strains. The prepared inoculums (0.2 ml/egg) were inoculated in 9-10 days-old embryonated eggs through allantoic route (Alexander and Senne, 2008) and incubated at 37°C and regularly candled for mortality of the embryos. The embryos that showed mortality within 36-48 hours post inoculation were kept for brief freezing at 4°C for 2 hrs and the allantoic fluid was harvested. The propagation of the NDV was confirmed by the haemagglutination inhibition test (HA-HI) and also by detection of F gene of NDV (Qin et al., 2008).
 
RNA extraction from clinical samples and molecular detection of NDV by reverse transcription-PCR (RT-PCR)
 
The total RNA was extracted from tissue samples (trachea, lungs, proventriculus, spleen and caecal tonsils) by using Trizol method (Chomczynski and Sacchi, 2006). Reverse transcription of RNA into cDNA was carried out by using cDNA synthesis kit (Fermentas Life Sciences, Canada) according to the manufacturer’s protocols. All the tissue samples were tested by RT-PCR for detection of the complete F gene of NDV by using the forward primer 5'-ATGGGCTCCAAACCTTCTAC-3' and reverse primer 5'-TTGTAGTGGCTCTCATC-3' (Qin et al., 2008). The general conditions for PCR were 95°C for 5 min, 32 cycles of 95°C for 1 min (denaturation), 52°C for 1 min (annealing) and 72°C for 2 min (extension), followed by 72°C for 10 min (final extension). PCR products were analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide. To rule out any co-infections with other respiratory diseases viruses, all the samples were also tested for infectious bronchitis virus (IBV) (Ji et al., 2011) and infectious laryngotracheitis (ILT) (Kirkpatrick et al., 2006).
 
Nucleotide sequencing and analysis
 
The 1662 bp products of complete F gene of Class II NDV amplified by RT-PCR were purified by using Thermo Scientific GeneJet Gel Extraction kit and cloned into pTZ57R/T vector using InsT/AcloneTM PCR product cloning kit (Fermentas Life Sciences, Canada). The recombinant plasmids containing the F gene were sequenced in DNA sequencing facility at Delhi University, South campus.
 
Phylogenetic analysis of complete fusion glycoprotein
 
The phylogenetic analyses of nucleotide sequences from complete F gene of three field isolates were performed as recommended by the recently updated unified phylogenetic classification system for NDV (Dimitrov et al., 2019). The dataset comprised 142 reference sequences (Table 1) spanning all 21 distinct genotypes of class II NDV (Dimitrov et al., 2019; Rajkhowa et al., 2023). The maximum-likelihood and Bayesian methods were used to construct the phylogenetic tree. The sequences were aligned using Muscle (MEGA7) and TrimAl (PhyloSuite v1.2.2) to remove spurious sequences or poorly aligned regions from a multiple sequence alignment (Zhang et al., 2020). The phylogenetic tree was inferred by Maximum-likelihood trees based on general time-reversible (GTR) model Tavare (1986), were constructed by using RaxML version 8.2.11 (Stamatakis, 2014) with 1000 bootstrap replicates. Trees were visualized using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree). 


 
Deduced amino acid sequence analysis of fusion proteins of the three field isolates were compared with three vaccine strains Mukteshwar, LaSota and B1 using MEGA7 (Kumar et al., 2016). Amino acid residues in hyper-variable region, fusion protein cleavage site, neutralizing epitopes, heptad repeat regions (HRb, HRc) and transmembrane domains of F protein sequences were compared.

Three distinct field outbreaks of NDV were confirmed from different commercial broiler farms located at Selesih, Zemabawk and Melthum areas in Aizawl district of Mizoram, during the study period from December 2021 to December 2022. In all three farms’ birds were vaccinated with LaSota strain (primary dose) and R2B strain of NDV (booster dose). The affected birds showed respiratory disease characterized by severe depression, cyanosis of combs and wattles, conjunctivitis and nasal discharges, coughing and respiratory rales accompanied by greenish diarrhoea. Detailed post-mortem examination of dead birds revealed petechial haemorrhages at the tip of proventricular glands, haemorrhagic ulceration on the caecal tonsils, severely congested or haemorrhagic trachea and lungs and enlarged, mottled spleen (Fig 1a to 1c). Microscopical examination consistently showed haemorrhagic tracheitis with sloughed-off mucosa, pneumonia characterized by severe congestion, oedema, haemorrhages and mononuclear infiltration in the parabronciolar parenchyma; lymphoid depletion with haemorrhages in spleen and caecal tonsils and haemorrhagic proventriculitis (Fig 1e and 1f). The outbreaks were confirmed by detection of the complete F gene of class II NDV in representative tissue samples (Spleen, Caecal tonsils, lungs and trachea).


 
Three field strains of the NDV were successfully isolated in 9-10 days old embryonated eggs and the NDV propagation was confirmed by the RT-PCR and HA-HI test. The embryos in the inoculated eggs died between 36-48 hours and showed dwarfing, curling and haemorrhages throughout the body surfaces (Fig 1d). 
 
The amplified 1662 bp product encompassing the complete F gene of NDV was sequenced, analyzed, submitted to GenBank and obtained the accession no. OQ427364, OQ427365 and OQ427366 (Table 1). The deduced amino acid analysis revealed presence of multi-basic amino acid residues at the fusion protein cleavage site between 112 and 116 and phenylalanine at position 117 (112RRQKRF117) were observed in all three field isolates confirming the isolates as velogenic strain (Table 2) (OIE, 2012). The comparison of amino acid sequences of fusion proteins of the three isolates with the vaccine strains Mukteshwar, LaSota and B1 showed 91.29% to 91.65%, 89.11% to 89.47%, 88.93% to 89.29% sequence identity respectively. Also, a comparison with genotype XIII strains that are recorded as circulating strains in mainland India, showed 89.87% to 93.67% sequence homology with the three field isolates. Although all three isolates showed similarity with the newly identified XXII genotype strains earlier identified from NER, India, two mutations at the positions N9V and P10S were observed in the hypervariable region (Table 2). No distinct variations were observed in the neutralizing epitope, heptad repeat regions HRa, HRb, HRc and transmembrane domains.


 
The phylogenetic analysis based on the complete F gene was carried out as per the recommendation put forth by the recently updated unified phylogenetic classification system for NDV (Dimitrov et al., 2019). The generated phylogenetic tree has depicted close grouping of all the three isolates with newly identified class II NDV genotype XXII and subgroup XXII.2 (Fig 2).


 
Our study has recorded outbreaks of ND that have resulted 90-100% mortality in the three affected commercial broiler farms. The outbreaks occurred despite the vaccination against the disease with LaSota strain followed by a booster dose with R2B strain. Consistent observation of haemorrhagic proventriculitis, enteritis, ulcerative and haemorrhagic caecal tonsils accompanied with haemorrhagic tracheitis and pneumonia have strongly suggested that the circulating strains are velogenic NDV (vvNDV). The outbreaks were confirmed by detection of complete F gene of NDV by RT-PCR assay (Qin et al., 2008). Further, the field strains were also isolated in 9-10 days old embryonated eggs.
 
The presence of multi-basic amino acid residues at the fusion protein cleavage site between 112 and 116 and phenylalanine at position 117 (112RRQKRF117) (Gowthaman et al., 2019; OIE, 2012) in all three field isolates have further confirmed the isolates as velogenic strain complementing our pathological findings (Table 2). The comparison of amino acid sequences of the entire fusion proteins of the three isolates with the vaccine strains Mukteshwar, LaSota and B1 revealed only 88.93% to 91.29% sequence identity suggesting independent evolution of the circulating NDV strains in Mizoram, India. Deduced amino acid analysis of all three isolates showed similarity with the newly identified XXII genotype strains earlier identified from NER, India, with additional two mutations at the positions N9V and P10S in the hypervariable region (Table 2).
 
The updated unified phylogenetic classification system for NDV (Dimitrov et al., 2019) has classified the class II NDV into at least 20 distinct genotypes (I to XXI). Following this classification, the field isolates from NER, India and Bangladesh were classified into a new genotype and designated as XXII with two subgroups XXII.1 and XXII.2 (Rajkhowa et al., 2023), while the NDV strains from rest of the mainland India were identified as genotype XIII (sub-genotype XIII.2.2.). Earlier studies (Khorajiya et al., 2015; Das and Kumar, 2017; Gowthaman et al., 2019; Mariappan et al., 2018) have also identified the NDV currently circulating and causing field outbreaks of ND from rest of the mainland India, as the genotype XIII. To understand the relation between the three field isolates, with all the 21 distinct genotypes of class II NDV strains, we have carried out the phylogenetic analysis based on the complete F gene (Dimitrov et al., 2019; Rajkhowa et al., 2023). The generated phylogenetic tree has clearly characterized the three field isolates into the newly identified genotype XXII and subgroup XXII.2 of class II NDV.  

NER, India, stands at high risk for transmission of transboundary diseases like ND, as it shares its boundaries with neighbouring countries namely China, Bhutan, Nepal, Myanmar and Bangladesh. The region lies at the junction of the Eastern Himalayas and the Indo-Burma biodiversity hotspot that provides a unique ecosystem for evolution and emergence of pathogens. Therefore, continuous surveillance and monitoring of infectious pathogens of livestock and birds in the region is of paramount interest for the control and prevention strategy of such diseases. The present study on field outbreaks of ND in vaccinated flock of birds has recorded severe clinical disease with high morbidity and mortality. The circulating field strains have been identified as velogenic strains and characterized in the genotype XXII, subgroup XXII.2 of class II NDV. The evolutionary evidence of a new genotype of NDV in the ecosystem of NER, India, warrants detailed studies on the whole genome of the identified genotype as well as animal experimentation to identify the possibility of neutralization escape variants.
 

We are thankful to the Dean, CVSc and AH, CAU(I), Selesih, Aizawl, Mizoram, for providing required infrastructure to carry out this research work.

No live animals were harmed during the present study conducted at any point of time.

 The authors declare no conflict of interest.