Explore the vast range of titles available.

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (10 4 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.

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.

Background The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. Results Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014–2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian ( n  = 2) and South Korean ( n  = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. Conclusions We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions.

Background Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. Methods An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. Results For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 10 1 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. Conclusion The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

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.

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 104.5 or 103.5 50% embryo infectious dose (EID50/egg) and chickens were challenged at 21 days post-hatch (DPH). In the second experiment, 103.5 EID50/egg of each vaccine was administered at 19 DOE, and chickens were challenged at 14 DPH. Chickens vaccinated with 103.5 EID50/egg of rZJ1*L-IL4R had hatch rates comparable to the group that received diluent alone, whereas other groups had significantly lower hatch rates. All vaccinated chickens survived challenge without displaying clinical disease, had protective hemagglutination inhibition titers, and shed comparable levels of challenge virus. The recombinant rZJ1*L-IL4R vaccine yielded lower post-vaccination mortality rates compared with the other in ovo NDV live vaccine candidates as well as provided strong protection post-challenge.

The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II.

Newcastle disease virus (NDV) has a wide avian host range and a high degree of genetic variability, and virulent strains cause Newcastle disease (ND), a worldwide concern for poultry health. Although NDV has been studied in Nigeria, genetic information about the viruses involved in the endemicity of the disease and the transmission that likely occurs at the poultry-wildlife interface is still largely incomplete. Next-generation and Sanger sequencing was performed to provide complete (n = 73) and partial genomic sequence data (n = 38) for NDV isolates collected from domestic and wild birds in Nigeria during 2002-2015, including the first complete genome sequences of genotype IV and subgenotype VIh from the African continent. Phylogenetic analysis revealed that viruses of seven different genotypes circulated in that period, demonstrating high genetic diversity of NDV for a single country. In addition, a high degree of similarity between NDV isolates from domestic and wild birds was observed, suggesting that spillovers had occurred, including to three species that had not previously been shown to be susceptible to NDV infection. Furthermore, the first spillover of a mesogenic Komarov vaccine virus is documented, suggesting a previous spillover and evolution of this virus. The similarities between viruses from poultry and multiple bird species and the lack of evidence for host adaptation in codon usage suggest that transmission of NDV between poultry and non-poultry birds occurred recently. This is especially significant when considering that some viruses were isolated from species of conservation concern. The high diversity of NDV observed in both domestic and wild birds in Nigeria emphasizes the need for active surveillance and epidemiology of NDV in all bird species.

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.