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Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99-100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3-98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000-2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

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.

Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry. ND causes heavy economic losses to the global poultry industry by decreasing the growth rate, decrease in egg production high morbidity and mortality. Although significant advances have been made in the vaccine development, outbreaks are reported in vaccinated birds. In this study, we report the damage caused by NDV infection in the pancreatic tissues of vaccinated and specific-pathogen-free chickens. The histopathological examination of the pancreas showed severe damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of the pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone and somatostatin were observed in NDV-infected chicken at three- and five- days post infection (DPI). A slight decrease in the plasma concentrations of insulin was noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection decreased the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Taken together, our findings highlight that NDV induces extensive tissue damage in the pancreas, decreases the activity and expression of pancreatic enzymes, and increases plasma corticosterone and somatostatin. These findings provide new insights that a defective pancreas may be one of the reasons for decreased growth performance after NDV infection in chickens.

Runting stunting syndrome (RSS) in commercial chickens has been reported worldwide, and although several studies have attempted to clarify the cause and describe the lesions, there are gaps in knowledge of the epidemiology, pathogenesis, and etiology. The study objective was to use commercial chicks naturally affected by RSS to describe the histologic changes of RSS in all segments of the small intestine in chicks of different ages and to identify viral gene sequences in affected chicks and their association with histologic lesions. Chicks lacking clinical signs but from the same houses and from unaffected houses were used as controls. The average weight of affected chicks was significantly lower than expected for their flocks. Macroscopically, the small intestines had paler serosa, with watery, mucoid, or foamy contents and poorly digested food. Histologic lesions were characterized by necrotic crypts, crypt dilation, and flattening of the crypt epithelium. Histomorphometry of the intestines revealed villous atrophy especially in the jejunum and ileum. Histologic changes in other organs were not observed. Random next-generation sequencing of total RNA extracted from formalin-fixed paraffin-embedded tissues detected avian nephritis virus, avian rotavirus, and picornavirus in jejunal segments from 7-day-old chicks. No viruses were detected in the jejunum of 1-day-old chicks. Detection of picornaviral reads was significantly associated ( P < .05) with histologic lesions of RSS. Sequence analysis of the picornavirus revealed genetic similarity with the genus Gallivirus. Using in situ hybridization for galliviral nucleic acid sequences, the signal was associated with crypt lesion severity, although signal was detected both in chicks with and without RSS.

Background: Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry, Newcastle disease (ND), which is particularly characterized by extensive pathologies in the digestive, respiratory and nervous systems. ND cause heavy economic losses to the world poultry industry by decreasing growth rate, decrease egg productions, mortality and morbidity. Although, significant advances have been made in the vaccine development, but outbreaks are reported in vaccinated birds leading decrease growth rate. Methods: In this study, we report the damage caused by the NDV infection in the pancreas of vaccinated as well specific pathogen free chickens. Results: The histopathological examination of the pancreas showed sever damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, and apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone, somatostatin, were observed in NDV infected chicks at 3 and 5-day post infection (DPI). Slight decrease in the plasma concentrations of the insulin were noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection has decreases the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Conclusion: Taken together, our findings highlight that NDV induces extensive tissue damage in pancreas, decrease the activity and expression of pancreatic enzymes and increase plasma corticosterone and somatostatin.

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 101 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. Electronic supplementary material The online version of this article (10.1186/s12985-018-1077-5) contains supplementary material, which is available to authorized users.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of infectious bronchitis is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. While serotyping by definition requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV and it is inefficient at detecting mixed isolates. This paper describes a MinION-based AmpSeq method that genetically typed IBV from clinical samples, including samples with multiple isolates. Total RNA was extracted from fifteen tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer’s instructions into a 1D MinION sequencing library, and sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in three of five samples containing two IBV lineages. Additionally, one sample contained three IBV lineages, and AmpSeq accurately detected two of the three. Strain identification, including detection of different strains from the same lineage, was also possible with this AmpSeq method. The results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

The present study was designed to investigate the toxico-pathological effect of cadmium (Cd) and its amelioration with silymarin (SL) and milk thistle (MT) in male Japanese quail (Coturnix japonica...

Background Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. Results Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. Conclusion These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype. Electronic supplementary material The online version of this article (10.1186/s12917-019-2053-z) contains supplementary material, which is available to authorized users.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is one of the most devastating diseases of the poultry. Despite the use of mass- and intense-vaccination regimens, the disease continues to sustain in many countries around the globe including Pakistan. This highlights the need to ascertain the characterization of field circulating strains at resolution that provides evidences of vaccine failures. In this study, a naturally occurring reassortant IBDV (referred as PK2) was identified and for the first time the full-genomic and pathogenic characterization of Pakistan strain was revealed. Genome analysis showed that the segment A of PK2 was derived from a very virulent IBDV (vvIBDV) whereas the segment B was acquired from another unique ancestor. Furthermore, infection of SPF chickens with PK2 revealed the potential of the virus in causing infection and inducing pathologies. Taken together, the present study provides useful data for further understanding of the popular and evolution of IBDV, which will helpfully support a better prevention and control of the disease in Pakistan.

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. A highly sensitive amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described using both egg-propagated virus and clinical samples. 1D MinION sequencing of barcoded NDV amplicons was performed on 33 egg-grown isolates, (23 unique lineages, including 15 different NDV genotypes), and from 15 clinical swab samples from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. For all egg-grown samples, NDV was detected and virulence and genotype were predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes, of which the MinION method detected both genotypes in four of those samples. Additionally, testing a dilution series of one NDV sample resulted in detection of NDV with a 50% egg infectious dose (EID50) as low as 101EID50/ml. This was accomplished in as little as 7 minutes of sequencing time, with a 98.37% sequence identity compared to the expected consensus. The high sensitivity, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for identification of NDV of different genotypes circulating worldwide. This general method will likely be applicable to other infections agents.