Clinical signs and gross lesions in naturally infected backyard chicken
Backyard poultry flock from which the samples were collected was not vaccinated against ND. Birds in the affected flock showed anorexia, depression, prostration, greenish diarrhea 3 days before the onset of mortality. The necropsy findings observed in few birds were pin point haemorrhages in the proventricular glands, segmental congestion in the intestinal mucosa and necrotic lesions in the caecal tonsils.
Isolation and characterization of AAvV-1
Tissue homogenates of dead backyard poultry resulted in peri- occipital haemorrhages and diffuse congestion in the inoculated dead embryos during the first passage. Of the forty eight cloacal swabs collected from the migratory birds, one sample from spot- billed pelican showed similar changes in the inoculated embryos during second passage. Amnio allantoic fluids (AAF) were collected 48h post inoculation and presence of AAvV-1 was confirmed by HA and HI assays employing polyclonal chicken antiserum raised against LaSota. AAvV-1 isolates confirmed by HI were further confirmed by RT-PCR targeting partial fusion gene encompassing FPCS. Both the isolates were classified as velogenic based on the MDT and ICPI values (Table 1).
The two isolates were plaque purified in confluent monolayers of chicken embryo fibroblast (CEF) cultures, stored at -800C and are hereby referred as AAvV/Chicken/SKLM-1/2018 (SKLM-1) and AAvV/Pelican/ Telineelapuram/2018 (MIG-9).
Genotype analysis
The amplified F and HN gene sequences of SKLM-1 and MIG-9 isolates were submitted to GenBank and are available under accession numbers: MT036311(SKLM-1/F); MZ270546 (SKLM-1/HN); MN901912 (MIG-9/F); MT909566 (MIG-9/HN).
The SKLM-1 and MIG-9 isolates were 100% identical to each other. The topology of the phylogenetic trees was found to be similar when constructed either based on full- length fusion or HN genes (Figure 1&2). Both the isolates were classified as members of genotype XIII of class II. Further analysis employing reference strains of sub- genotypes of genotype XIII (1.1, 1.2, 2.1, 2.2) showed that the isolates clustered closely with viruses belonging to sub- genotype XIII 2.2 isolates. Based on the fusion gene sequences, these isolates are closely related and have 99.2% identity with Avian Avula virus-1 isolate 410/16A (GenBank ID MF422129), 98.3% identity with Avian Avula virus-1 isolate 96-15 (GenBank ID MF422125) and 97.8% with AAVV isolate D162 (GenBank ID KX242342), which are all placed under the same subgenotype. Further, isolates like NDV strain Nagpur (GenBank ID KP089979), NDV/16/GODHRA/03/2013 (GenBank ID KM056344), NDV40/SARSA/04/2013 (GenBank ID KM056348), NDV/NAGPUR/04/11 (GenBank ID KX372708) are comparatively less identical with a divergence value of more than 5% although they were also included in the same genotype XIII 2.2. Based on HN gene sequences, AAvV/Pelican/ Telineelapuram/2018 isolate have 99.7 % identity with Avian Avula virus-1 isolate 410/16A (GenBank ID MF422129) and AAvV/Chicken/SKLM-1/2018 have 98.43% identity with Avian Avula virus-1 isolate 96-15 (GenBank ID MF422125).
The deduced amino acid sequence of the fusion protein of both the isolates showed only a divergence of 0.8% with sub- genotype XIII 2.2 isolates, while the divergence with current vaccine strains ranged from 10-12.9%. The deduced amino acid sequence of the HN protein showed a divergence of 0.3- 1.57% with sub- genotype XIII 2.2 isolates and 11.3- 14.5% with current vaccine strains.
Analysis of fusion gene sequences
Fusion genes of the two isolates were found to have a cds of 1662 nucleotides coding for 553 amino acids. Amino acid sequence at the FPCS cleavage site motif 112R-R-R-K-R-F117 has more than two pairs of basic amino acids and a Phenylalanine in the N terminus of the F1 protein (Table 1) typical for velogenic isolates. Table 2 shows the amino acid substitutions within the fusion peptide, heptad repeat regions (HRa, HRb, HRc and HRd) and transmembrane domains of the study isolates in comparision to the vaccine strains. Several amino acid substitutions were observed in these domains which are also noted in the reference strains of genotype XIII sub- genotype 2.2. Of the eight transmembrane domains in the fusion protein, six domains located at positions 15-25, 118-131, 120-128, 266-269, 429-432 and 499-525 were unaltered, while several substitutions were noted in the domains, 14-27 and 501- 523. Six potential N-glycosylation sites (Asn-X-Ser/Thr or N-X-S/T, where X present any amino acid except aspartic acid or proline) at positions 85NRT87, 191NNT193, 366NTS368, 447NIS449, 471NNS473 and 541NNT543 were unaltered in both the study isolates.
Amino acid substitutions within the hypervariable region and neutralizing epitopes of the isolates under study were compared with the vaccine strains (Table 3). Several unique substitutions were observed in the hypervariable region of the study isolates (R4K, A11I, M13L, M14G, T16I, I17T, A20M, V22I, P28L, A29T and L69I). M14G substitution is unique to the present isolates and is not identified even in the reference strains of genotype XIII sub- genotype 2.2. Interestingly, hypervariable region of the study isolates were similar to Mukteshwar strain at amino acid positions 8,9,13,28,29 and 31 and to other vaccine strains at positions viz., 10, 25, 26, 27 and 30. Neutralizing epitope sequences critical for structure and function of the fusion protein were similar to that of the vaccine strains.
Analysis of HN gene sequences
HN genes of the two isolates were found to have a cds of 1716 nucleotides coding for 571 amino acids. Three transmembrane domains at positions 24-47, 25-45 and 557-563, a sialic acid binding site at position 234-239 and 13 Cystine (C) residues at positions 123, 172, 186, 196, 238, 247, 251, 344, 455, 461, 465, 531 and 542 were recognised in the HN proteins of either of the isolates. The Cysteine (C) residue at position 123 was present in R2B and Mukteshwar vaccine strains also, while it has been replaced by Tryptophan (W) in LaSota, Komarov and B1.
The HN proteins of the isolates were also found to have six glycosylation sites at positions 119, 341, 433, 481, 508 and 538. As compared to the vaccine strains, the study isolates have amino acid substitutions at positions G75S, N77G, V81I and I84V in heptad repeat region A domain and T101S and T102I substitutions in heptad repeat region B domain. Five neutralizing epitopes have been recognised in the HN protein of which, two epitopes viz., site 23 (193-201) and site 1 and 14 (345-355) remained unaltered, while three epitopes viz., site 12 (494), site 2 and 12 (513-521) and site 2 (569) presented numerous substitutions (Table 4). Three amino acids at positions, 401E, 416R and 526Y characterized by functional triarginyl cluster considered essential for receptor binding and neuraminidase activity are conserved in the study isolates. HN protein motifs responsible for hemagglutinating activity, viz., 234NRKSCSV/I/L240, 314FPVYGGL/V/M320 and 399GAEGRIL/V/I405 remained unaltered in the study isolates.
Pathogenicity assay in three- week old chicken
Infection of three- week old chicken with the SKLM-1 and MIG-9 AAvV-1 isolates resulted in virulent ND with 100% mortality. Clinical signs, gross and histopathological lesions induced by either of the isolates appeared similar and indicated that they caused systemic infection in chicks and replicated quickly in multiple tissues. Birds inoculated with either of the isolates, had evident clinical disease (depression, reluctancy to move, open mouthed breathing, mild conjunctivitis, ruffled feathers and greenish diarrhoea) at 2 dpi which became more severe until death at 4 or 5 dpi. Also, neurological signs like ataxia, twitching, head tremors were observed hours before death. Birds exhibiting severe clinical signs were euthanised to perform necropsy. Gross lesions were found widespread throughout different tissues. Most frequent necropsy findings include hemorrhages in the proventriculus and caecal tonsils, swollen kidneys, tracheal hemorrhages and splenomegaly associated with mottling (Fig.3). The sentinel birds also started exhibiting similar clinical signs as the directly inoculated birds 5 days post exposure progressing to severe clinical signs by 7 days post exposure. Hundred percent mortality was observed in all the infected and sentinel birds. Control birds appeared clinically normal throughout the observation period.
Histological lesions were analysed in the tissues of infected, sentinel and control birds. No major differences were noticed in the birds infected with either of the viruses and in the sentinel birds. In spleen, lesions varied from vacoular changes to necrosis of the lymphoid tissue in the white pulp characterized by lymphocytolysis and lymphoid depletion. Heart revealed pericarditis and myocarditis characterized by degeneration of myocardium, infiltration of heterophils, fibrin, fibroblasts and mononuclear cells in the pericardium and myocardium. In the small intestines, lesions varied from catarrhal enteritis characterized by severe goblet cell hyperplasia to fusion, necrosis of villi and desquamation of epithelium into lumen. Lesions observed in the trachea during acute phase of respiratory disease were haemorrhages and vacoular changes in the tracheal mucosa, syncytia formation and denudation of tracheal epithelium. Lungs revealed severe congestion and consolidation of parenchyma, denudation and exudation of bronchiolar epithelium. In the kidneys, lesions included nephrosis characterized by degeneration to severe tubular necrosis and interstitial nephritis (Fig.4). No specific lesions were observed in the control group.