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The hypothesis that host adaptation in virulent Newcastle disease viruses (NDV) has been accompanied by virulence modulation is reviewed here. Historical records, experimental data, and phylogenetic analyses from available GenBank sequences suggest that currently circulating NDVs emerged in the 1920–1940′s from low virulence viruses by mutation at the fusion protein cleavage site. These viruses later gave rise to multiple virulent genotypes by modulating virulence in opposite directions. Phylogenetic and pathotyping studies demonstrate that older virulent NDVs further evolved into chicken-adapted genotypes by increasing virulence (velogenic-viscerotropic pathotypes with intracerebral pathogenicity indexes [ICPIs] of 1.6 to 2), or into cormorant-adapted NDVs by moderating virulence (velogenic–neurotropic pathotypes with ICPIs of 1.4 to 1.6), or into pigeon-adapted viruses by further attenuating virulence (mesogenic pathotypes with ICPIs of 0.9 to 1.4). Pathogenesis and transmission experiments on adult chickens demonstrate that chicken-adapted velogenic-viscerotropic viruses are more capable of causing disease than older velogenic-neurotropic viruses. Currently circulating velogenic–viscerotropic viruses are also more capable of replicating and of being transmitted in naïve chickens than viruses from cormorants and pigeons. These evolutionary virulence changes are consistent with theories that predict that virulence may evolve in many directions in order to achieve maximum fitness, as determined by genetic and ecologic constraints.

Direct-targeted next-generation sequencing (tNGS), with its undoubtedly superior diagnostic capacity over real-time PCR (RT-PCR), and direct-non-targeted NGS (ntNGS), with its higher capacity to identify and characterize multiple agents, are both likely to become diagnostic methods of choice in the future. tNGS is a rapid and sensitive method for precise characterization of suspected agents. ntNGS, also known as agnostic diagnosis, does not require a hypothesis and has been used to identify unsuspected infections in clinical samples. Implemented in the form of multiplexed total DNA metagenomics or as total RNA sequencing, the approach produces comprehensive and actionable reports that allow semi-quantitative identification of most of the agents present in respiratory, cloacal, and tissue samples. The diagnostic benefits of the use of direct tNGS and ntNGS are high specificity, compatibility with different types of clinical samples (fresh, frozen, FTA cards, and paraffin-embedded), production of nearly complete infection profiles (viruses, bacteria, fungus, and parasites), production of “semi-quantitative” information, direct agent genotyping, and infectious agent mutational information. The achievements of NGS in terms of diagnosing poultry problems are described here, along with future applications. Multiplexing, development of standard operating procedures, robotics, sequencing kits, automated bioinformatics, cloud computing, and artificial intelligence (AI) are disciplines converging toward the use of this technology for active surveillance in poultry farms. Other advances in human and veterinary NGS sequencing are likely to be adaptable to avian species in the future.

U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

Metagenomics offers the potential to replace and simplify classical methods used in the clinical diagnosis of human and veterinary infectious diseases. Metagenomics boasts a high pathogen discovery rate and high specificity, advantages absent in most classical approaches. However, its widespread adoption in clinical settings is still pending, with a slow transition from research to routine use. While longer turnaround times and higher costs were once concerns, these issues are currently being addressed by automation, better chemistries, improved sequencing platforms, better databases, and automated bioinformatics analysis. However, many technical options and steps, each producing highly variable outcomes, have reduced the technology’s operational value, discouraging its implementation in diagnostic labs. We present a case for utilizing non-targeted RNA sequencing (NT-RNA-seq) as an ideal metagenomics method for the detection of infectious disease-causing agents in humans and animals. Additionally, to create operational value, we propose to identify best practices for the “core” of steps that are invariably shared among many human and veterinary protocols. Reference materials, sequencing procedures, and bioinformatics standards should accelerate the validation processes necessary for the widespread adoption of this technology. Best practices could be determined through “implementation research” by a consortium of interested institutions working on common samples.

Background Avian avulavirus (commonly known as avian paramyxovirus-1 or APMV-1) can cause disease of varying severity in both domestic and wild birds. Understanding how viruses move among hosts and geography would be useful for informing prevention and control efforts. A Bayesian statistical framework was employed to estimate the evolutionary history of 1602 complete fusion gene APMV-1 sequences collected from 1970 to 2016 in order to infer viral transmission between avian host orders and diffusion among geographic regions. Ancestral states were estimated with a non-reversible continuous-time Markov chain model, allowing transition rates between discrete states to be calculated. The evolutionary analyses were stratified by APMV-1 classes I ( n  = 198) and II ( n  = 1404), and only those sequences collected between 2006 and 2016 were allowed to contribute host and location information to the viral migration networks. Results While the current data was unable to assess impact of host domestication status on APMV-1 diffusion, these analyses supported the sharing of APMV-1 among divergent host taxa. The highest supported transition rate for both classes existed from domestic chickens to Anseriformes (class I:6.18 transitions/year, 95% highest posterior density (HPD) 0.31–20.02, Bayes factor (BF) = 367.2; class II:2.88 transitions/year, 95%HPD 1.9–4.06, BF = 34,582.9). Further, among class II viruses, domestic chickens also acted as a source for Columbiformes (BF = 34,582.9), other Galliformes (BF = 34,582.9), and Psittaciformes (BF = 34,582.9). Columbiformes was also a highly supported source to Anseriformes (BF = 322.0) and domestic chickens (BF = 402.6). Additionally, our results provide support for the diffusion of viruses among continents and regions, but no interhemispheric viral exchange between 2006 and 2016. Among class II viruses, the highest transition rates were estimated from South Asia to the Middle East (1.21 transitions/year; 95%HPD 0.36–2.45; BF = 67,107.8), from Europe to East Asia (1.17 transitions/year; 95%HPD 0.12–2.61; BF = 436.2) and from Europe to Africa (1.06 transitions/year, 95%HPD 0.07–2.51; BF = 169.3). Conclusions While migration appears to occur infrequently, geographic movement may be important in determining viral diversification and population structure. In contrast, inter-order transmission of APMV-1 may occur readily, but most events are transient with few lineages persisting in novel hosts.

Highly pathogenic avian influenza (HPAI) virus subtype H7N3 has been circulating in poultry in Mexico since 2012 and vaccination has been used to control the disease. In this study, eight Mexican H7N3 HPAI viruses from 2015-2017 were isolated and fully sequenced. No evidence of reassortment was detected with other avian influenza (AI) viruses, but phylogenetic analyses show divergence of all eight gene segments into three genetic clusters by 2015, with 94.94 to 98.78 percent nucleotide homology of the HA genes when compared to the index virus from 2012. The HA protein of viruses from each cluster showed a different number of basic amino acids (n = 5-7) in the cleavage site, and six different patterns at the predicted N-glycosylation sites. Comparison of the sequences of the Mexican lineage H7N3 HPAI viruses and American ancestral wild bird AI viruses to characterize the virus evolutionary dynamics showed that the nucleotide substitution rates in PB2, PB1, PA, HA, NP, and NS genes greatly increased once the virus was introduced into poultry. The global nonsynonymous and synonymous ratios imply strong purifying selection driving the evolution of the virus. Forty-nine positively selected sites out of 171 nonsynonymous mutations were identified in the Mexican H7N3 HPAI viruses, including 7 amino acid changes observed in higher proportion in North American poultry origin AI viruses isolates than in wild bird-origin viruses. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further improving control measures including vaccination.

Background Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. Methods In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform. Results Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25–30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2–3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. Conclusions This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.

Strains of Newcastle disease virus (NDV) can be separated into genotypes based on genome differences even though they are antigenically considered to be of a single serotype. It is widely recognized that an efficacious Newcastle disease (ND) vaccine made with any NDV does induce protection against morbidity and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from infection and viral shed from such a challenge. Vaccines prepared from ND viruses corresponding to five different genotypes were compared to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced and the amount of challenge virus shed. Six groups of 4-week-old specific pathogen-free Leghorn chickens were given oil-adjuvanted vaccines prepared from one of five different inactivated ND viruses including strains B1, Ulster, CA02, Pigeon84, Alaska196, or an allantoic fluid control. Three weeks post-vaccination, serum was analyzed for antibody content using a hemagglutination inhibition assay against each of the vaccine antigens and a commercial NDV ELISA. After challenge with virulent CA02, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for virus shedding. All vaccines except for the control induced greater than 90% protection to clinical disease and mortality. The vaccine homologous with the challenge virus reduced oral shedding significantly more than the heterologous vaccines. NDV vaccines formulated to be phylogenetically closer to potential outbreak viruses may provide better ND control by reducing virus transmission from infected birds.

Nontargeted metagenomic surveillance of the poultry enteric virome reveals underrecognized threats to poultry health and productivity in intensive production systems. In South Asia, avian rotavirus A (AvRV-A) and avian orthoreovirus (ARV) are frequently detected in broilers by conventional diagnostics, whereas chicken megrivirus genotype C (ChMeV-C) is often identified through metagenomic surveillance. Often present in both clinical disease and coinfections, these viruses may impair gut function, immune responses, and growth performance, yet their genomic diversity and evolutionary dynamics in poultry remain poorly characterized. Here, we report complete genomes of AvRV-A, ARV, and ChMeV-C strains co-detected via nontargeted metagenomic next-generation sequencing (ntNGS) in a pooled cloacal sample comprising 150 commercial broiler chickens (19 and 33 days old) collected from three commercial farms in Kamrup Rural District, Assam, Northeast India. Despite routine vaccination, all three flocks experienced > 10% mortality, poor weight gain, and postmortem lesions including pale kidneys and hepatomegaly. Phylogenetic analyses revealed segmental clustering in ARV and AvRV-A consistent with reassortment-driven divergence, though not supported by detectable recombination, while ChMeV-C clustered within a distinct C1 sublineage, suggesting intercontinental lineage connectivity and highlighting the need to expand regional genomic baseline data. We also identified nonsynonymous single nucleotide polymorphisms in several key viral proteins, including RNA-dependent RNA polymerases (VP1 of AvRV-A, λB of ARV, and 3D of ChMeV-C), capsid proteins (VP2 and VP7 of AvRV-A, λA and σB of ARV, and VP0 and VP1 of ChMeV-C), and replication-associated nonstructural proteins. These findings expand the genomic baseline for poultry enteric viruses in South Asia, reveal novel polymorphic signatures, and underscore the value of ntNGS-based metagenomic surveillance in virus detection, diversity monitoring, and informing vaccine and biosecurity strategies.

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.