MOLECULAR DETECTION AND CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS STRAINS FROM POULTRY
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Date
2021-10
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College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
Abstract
Newcastle disease is a highly transmissible and acute fatal disease of poultry caused by virulent strains of Avian paramyxovirus type 1 (APMV-1) which is commonly known as the Newcastle disease virus (NDV). Avian paramyxovirus type 1 exhibit great variation in their pathogenicity and the severity of the disease produced varies with the host species and the strain of virus involved. Newcastle disease can have devastating effects on the poultry industry due to the high morbidity and mortality associated with virulent strains of the virus.
A study was undertaken to detect and characterize different NDV strains circulating among the native poultry population. To investigate the presence of NDV in clinically suspected backyard chickens, a total of 289 tissue samples were collected from 74 birds at necropsy from nine districts of Assam and tested using haemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR). Out of the 289 tissue samples, 24.57 % and 47.05 % samples were found to be positive for NDV in HI assay and RT-PCR respectively. Of the 74 clinically suspected chickens 52.70 % birds were found to be positive for NDV in HI assay while 91.89 % birds were found to be positive for NDV in RT-PCR. Among the different tissue samples tested for presence of NDV, a significantly higher number of tissue samples from spleen, trachea, lung, proventriculus and caecal tonsil tested positive for NDV irrespective of the test used. To detect NDV in apparently healthy chickens, 186 numbers of oropharyngeal swabs and 146 numbers of cloacal swabs were tested using HI assay and RT-PCR. Of the 186 oropharyngeal swabs tested, 24.57 % and 15.05 % swab samples were found to be positive for NDV in HI assay and RT-PCR respectively. Further, out of 146 cloacal swabs tested, 6.16 % and 21.23 % swab samples were found to be positive for NDV in HI assay and RT-PCR respectively.
A total of 18 tissue samples, identified as NDV positive using HI assay and RT-PCR, were processed for isolation of NDV using SPF embryonated chicken eggs (ECEs) of 9-11 days of incubation. The presence of the virus in the allantoic fluid of the inoculated ECEs was confirmed by two RT-PCR techniques, one of which targeted a 767 bp sequence of the F gene while the other targeted a 426 bp sequence of the HN gene of NDV. NDV from all the 18 tissue samples (100 %) was detected in the allantoic fluid of ECEs using the RT-PCR techniques.
Six representative NDV isolates were sequenced by outsourcing and subjected to phylogenetic study. The consensus sequences of the isolates were subjected to multiple sequence alignment with reference sequences from GenBank databases. A phylogenetic tree was then constructed where five of the isolates clustered with genotype XIII of Class II NDV while one clustered with genotype II of Class II NDV cluster. Evaluation of the amino acid composition of the F0 cleavage site revealed the presence of the consensus sequence 112R-R-Q-K-R-F117 in case of the five genotype XIII isolates whereas the genotype II NDV isolate possessed the sequence 112G-R-Q-G-R-L117 at the F0 cleavage site of the fusion gene. Thus, five isolates from the present study were identified as virulent NDV strains while one isolate was identified as an avirulent strain.
A multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) was standardized for simultaneous detection and differentiation of different pathotypes of NDV. Three specific oligonucleotide primers were used in the mRT-PCR for amplification of the target sequences of the F gene of NDV. The pathotypes of NDV was differentiated based on the products generated by the mRT-PCR. A product in size of 364 bp was obtained in case of the lentogenic strains while for mesogenic strains two
products in size of 364 bp and 204 bp were generated. In case of velogenic strains only one product in size of 204 bp was generated. All the 18 NDV isolates from the present study was characterized using the mRT-PCR. Out of the eighteen isolates one isolate was identified as a lentogenic and two were identified as mesogenic. The other 15 isolates were identified as velogenic strains. A standard RFLP technique was also used to validate the results of the mRT-PCR. The RFLP involved digestion of a RT-PCR amplified 363 bp fusion gene product by HinfI restriction enzyme. The virulent and avirulent strains of NDV were differentiated based on the HinfI digestion patterns exhibited on 3 % agarose gels. The results of pathotyping obtained using RFLP analysis, corroborated the results of the mRT-PCR.
A local isolate of NDV (AS/KM/CG 01) from non-vaccinated backyard chicken was subjected to complete genome sequencing by outsourcing. Evaluation of the genomic data revealed that the genome of the NDV isolate AS/KM/CG 01 consists of six genes arranged in tandem that encodes for six structural proteins namely, the nucleocapsid protein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin-neuraminidase protein (HN), and the polymerase protein (L). The isolate possessed a genome of approximately 15 kb. Evaluation of the F0 cleavage site within the fusion gene of the isolate revealed the presence of the consensus amino acid sequence 112G-R-Q-G-R-L117 which is typical of lentogenic strains of NDV. Phylogenetic study revealed that the NDV isolate belong to genotype II of class II NDV cluster. It was also found that the isolate has close relationship with previously reported genotype II NDV isolates from India and China. The isolate also showed more than 97 % homology with NDV vaccine strain LaSota.
The study was summarized with the findings that genotype XIII NDV of class II cluster is predominant among the poultry flocks of Assam. Detection of genotype II NDV of class II closely related to NDV vaccine strain LaSota in the present study suggests a possible spillover of vaccine-type viruses from vaccinated poultry or feral avian reservoirs to non-vaccinated backyard chickens. However further studies are needed on this aspect.