Virulent Newcastle disease virus genotypes V.3, VII.2, and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania.

Oropharyngeal (OP) and cloacal (CL) swabs from 2049 adult backyard chickens collected at 12 live bird markets, two each in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga regions of Tanzania were screened for Newcastle disease virus (NDV) using reverse transcription real-time PCR (rRT-PCR). The virus was confirmed in 25.23% of the birds (n = 517; rRT-PCR CT ≤ 30), with the highest positivity rates observed in birds from Dar es Salaam region with higher prevalence during the dry season (September-November 2018) compared to the rainy season (January and April-May 2019). Next-generation sequencing of OP/CL samples of 20 out of 32 birds that had high amounts of viral RNAs (CT ≤ 25) resulted in the assembly of 18 complete and two partial genome sequences (15,192 bp and 15,045-15,190 bp in length, respectively) of NDV sub-genotypes V.3, VII.2 and XIII.1.1 (n = 1, 13 and 4 strains, respectively). Two birds had mixed NDV infections (V.3/VII.2 and VII.2/XIII.1.1), and nine were coinfected with viruses of families Astroviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, Pneumoviridae, and Reoviridae. Of the coinfecting viruses, complete genome sequences of two avastroviruses (a recombinant chicken astrovirus antigenic group-Aii and avian nephritis virus genogroup-5) and two infectious bronchitis viruses (a turkey coronavirus-like recombinant and a GI-19 virus) were determined. The fusion (F) protein F1/F2 cleavage sites of the Tanzanian NDVs have the consensus motifs 112 RRRKR↓F 117 (VII.2 strains) and 112 RRQKR↓F 117 (V.3 and XIII.1.1 strains) consistent with virulent virus; virulence was confirmed by intracerebral pathogenicity index scores of 1.66-1.88 in 1-day-old chicks using nine of the 20 isolates. Phylogenetically, the complete F-gene and full genome sequences regionally cluster the Tanzanian NDVs with, but distinctly from, other strains previously reported in eastern and southern African countries. These data contribute to the understanding of NDV epidemiology in Tanzania and the region.

Genome Sequencing and Characterization of an Avian Orthoavulavirus 1 VG/GA-like Isolate with a Unique Fusion Cleavage Site Motif.

A complete genome sequence of a VG/GA -like strain of avian orthoavulavirus 1 (AOAV-1) was identified by nontargeted next-generation sequencing of an oropharyngeal swab sample collected from a carcass of a 12-mo-old backyard chicken. The isolate has a fusion (F) protein cleavage site motif consistent with a low virulent AOAV-1, but it has a unique motif with phenylalanine at position 117 (112G-R-Q-G-R↓F117), which is typical for virulent AOAV-1 strains. The one nucleotide difference at the cleavage site compared to other low-virulence viruses made the isolate detectable by F-gene-specific real-time reverse transcription-PCR (rRT-PCR) developed as a diagnostic test to specifically detect virulent strains. The mean death time determined in eggs and intracerebral pathogenicity index determined in chickens classified the isolate as lentogenic. This is the first report of a lentogenic VG/GA-like virus with a phenylalanine residue at position 117 of the F protein cleavage site in the United States. In addition to concern for potential pathogenic shift of the virus through additional changes at the cleavage site, our finding warrants increased awareness of diagnosticians of potential false positive F-gene rRT-PCR tests.

Secuenciación y caracterización del genoma de un aislado similar a VG/GA del ortoavulavirus aviar 1 con un motivo único en el sitio de disociación del gene de fusión. Se identificó una secuencia genómica completa de una cepa similar a la cepa Villegas-Glisson/Universidad de Georgia (VG/GA) del ortoavulavirus aviar 1 (AOAV-1) mediante secuenciación no dirigida de nueva generación de una muestra de hisopo orofaríngeo recolectada de una gallina muerta de traspatio de 12 meses. El aislado tiene un motivo en el sitio de disociación de la proteína de fusión (F) consistente con un ortoavulavirus aviar de baja virulencia, pero tiene un motivo único con fenilalanina en la posición 117 (112G-R-Q-G-R↓F117), que es típico para cepas virulentas del AOAV-1. La diferencia de un nucleótido en el sitio de escisión en comparación con otros virus de baja virulencia hizo que el aislado fuera detectable mediante transcripción reversa y PCR en tiempo real en tiempo real específica del gene F (rtRT-PCR) desarrollada como una prueba de diagnóstico para detectar específicamente a las cepas virulentas. El tiempo medio de muerte determinado en huevos y el índice de patogenicidad intracerebral determinado en pollos clasificaron al aislado como lentogénico. Este es el primer informe en los Estados Unidos de un virus lentogénico similar a VG/GA con un residuo de fenilalanina en la posición 117 del sitio de disociación de la proteína F. Además de la preocupación por el posible cambio patogénico del virus a través de cambios adicionales en el sitio de disociación, nuestro contribuye con un mayor conocimiento por parte del personal de diagnóstico acerca de posibles falsos positivos en las pruebas rtRT-PCR del gene F.

Unique Variants of Avian Coronaviruses from Indigenous Chickens in Kenya.

The avian gamma-coronavirus infectious bronchitis virus (AvCoV, IBV; Coronaviridae family) causes upper respiratory disease associated with severe economic losses in the poultry industry worldwide. Here, we report for the first time in Kenya and the Eastern African region two novel AvCoVs, designated IBV/ck/KE/1920/A374/2017 (A374/17) and AvCoV/ck/KE/1922/A376/2017 (A376/17), inadvertently discovered using random nontargeted next-generation sequencing (NGS) of cloacal swabs collected from indigenous chickens. Despite having genome organization (5'UTR-[Rep1a/1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR), canonical conservation of essential genes and size (~27.6 kb) typical of IBVs, the Kenyan isolates do not phylogenetically cluster with any genotypes of the 37 IBV lineages and 26 unique variants (UVs). Excluding the spike gene, genome sequences of A374/17 and A376/17 are only 93.1% similar to each other and 86.7-91.4% identical to genomes of other AvCoVs. All five non-spike genes of the two isolates phylogenetically cluster together and distinctly from other IBVs and turkey coronaviruses (TCoVs), including the indigenous African GI-26 viruses, suggesting a common origin of the genome backbone of the Kenyan isolates. However, isolate A376/17 contains a TCoV-like spike (S) protein coding sequence and is most similar to Asian TCoVs (84.5-85.1%) compared to other TCoVs (75.6-78.5%), whereas isolate A374/17 contains an S1 gene sequence most similar to the globally distributed lineage GI-16 (78.4-79.5%) and the Middle Eastern lineage GI-23 (79.8-80.2%) viruses. Unanswered questions include the actual origin of the Kenyan AvCoVs, the potential pathobiological significance of their genetic variations, whether they have indeed established themselves as independent variants and subsequently spread within Kenya and to the neighboring east/central African countries that have porous live poultry trade borders, and whether the live-attenuated Mass-type (lineage GI-1)-based vaccines currently used in Kenya and most of the African countries provide protection against these genetically divergent field variants.

Surveillance and Genetic Characterization of Virulent Newcastle Disease Virus Subgenotype V.3 in Indigenous Chickens from Backyard Poultry Farms and Live Bird Markets in Kenya.

Kenyan poultry consists of ~80% free-range indigenous chickens kept in small flocks (~30 birds) on backyard poultry farms (BPFs) and they are traded via live bird markets (LBMs). Newcastle disease virus (NDV) was detected in samples collected from chickens, wild farm birds, and other domestic poultry species during a 2017-2018 survey conducted at 66 BPFs and 21 LBMs in nine Kenyan counties. NDV nucleic acids were detected by rRT-PCR L-test in 39.5% (641/1621) of 1621 analyzed samples, of which 9.67% (62/641) were NDV-positive by both the L-test and a fusion-test designed to identify the virulent virus, with a majority being at LBMs (64.5%; 40/62) compared to BPFs (25.5%; 22/62). Virus isolation and next-generation sequencing (NGS) on a subset of samples resulted in 32 complete NDV genome sequences with 95.8-100% nucleotide identities amongst themselves and 95.7-98.2% identity with other east African isolates from 2010-2016. These isolates were classified as a new sub-genotype, V.3, and shared 86.5-88.9% and 88.5-91.8% nucleotide identities with subgenotypes V.1 and V.2 viruses, respectively. The putative fusion protein cleavage site (113R-Q-K-R↓F 117) in all 32 isolates, and a 1.86 ICPI score of an isolate from a BPF chicken that had clinical signs consistent with Newcastle disease, confirmed the high virulence of the NDVs. Compared to genotypes V and VI viruses, the attachment (HN) protein of 18 of the 32 vNDVs had amino acid substitutions in the antigenic sites. A time-scaled phylogeographic analysis suggests a west-to-east dispersal of the NDVs via the live chicken trade, but the virus origins remain unconfirmed due to scarcity of continuous and systematic surveillance data. This study reveals the widespread prevalence of vNDVs in Kenyan backyard poultry, the central role of LBMs in the dispersal and possibly generation of new virus variants, and the need for robust molecular epidemiological surveillance in poultry and non-poultry avian species.