Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania

Abstract

Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.

Keywords: East Africa; genotypes; newcastle disease virus (NDV); phylogenetic analyses; poultry; tanzania.

Figure 1

Clinical signs and necropsy findings in domestic chickens with Newcastle disease (ND). Chickens in a backyard poultry farm where suspected ND cases occurred, showing (A,B) pinpoint petechial hemorrhage on the proventriculus glands (indicated by a red arrowhead).

Figure 2

Phylogenetic analyses based on the partial F gene sequences of NDV strains. The 40 nucleotide sequences described in this study are marked with ● and labeled in red (subgenotype VII.2) or green (subgenotype XIII.1.1). Sixty-five nucleotide sequences representing all class I (including five unclassified viruses of class I, UNCL 9 to 13) and II subgenotypes were involved in this analysis. Phylogenic relationships using 1000 bootstraps were determined with MEGA X [39] using ClustalW alignment algorithm and maximum likelihood method for phylogenetic tree reconstruction. The evolutionary history was inferred by using the maximum likelihood and general time-reversible (GTR) model with a discrete gamma distribution (+G), allowing for invariant sites (+I) [45]. Codon positions included in the analysis were 1st + 2nd + 3rd + noncoding.

Figure 3

Phylogenetic analyses based on the partial F gene nucleotide sequences of NDV strains. The 39 genotype VII nucleotide sequences described in this study are marked with ● and labeled in red. Forty-one nucleotide sequences representing all genotype VII subgenotypes and 2 outliers (JX915243 from genotype XVI and AF048763 from genotype VIII) were included in this analysis. Phylogenic relationship using 1000 bootstraps was determined with MEGA X [39] using ClustalW alignment algorithm and maximum likelihood method for tree construction. The evolutionary history was inferred by using the maximum likelihood and general time-reversible (GTR) model with a discrete gamma distribution (+G), allowing for invariant sites (+I) [45]. Codon positions included in the analysis were 1st + 2nd + 3rd + noncoding.

Figure 4

Phylogenetic analyses based on the partial F gene nucleotide sequences of NDV strains. The single genotype XIII nucleotide sequence described in this study is marked with ● and labeled green. Thirty-two nucleotide sequences representing all genotype XIII subgenotypes and 4 outliers (AY741404 from genotype IV, EF201805 from genotype III, JX915243 from genotype XVI, and AF048763 from genotype VIII) were included in this analysis. Phylogenic relationship using 1000 bootstraps was determined with MEGA X [39] using ClustalW alignment algorithm and maximum likelihood method for tree construction. The evolutionary history was inferred by using the maximum likelihood and general time-reversible (GTR) model with a discrete gamma distribution (+G), allowing for invariant sites (+I) [45]. Codon positions included in the analysis were 1st + 2nd + 3rd + noncoding.

Figure 5

Pairwise identities of the 39 genotype VII strains’ partial fusion protein sequences that were aligned by ClustalW and shown by the software Sequence Demarcation Tool (SDT).

Figure 6

Sequence Demarcation Tool (SDT) software’s display of the pairwise identities plot of fusion protein sequences aligned by ClustalW for our sole genotype XIII strain.